Chem Soc Rev€¦ · This ournal is ' The Royal Society of Chemistry 2019 Chem.Soc.Rev. (i) generalize across DNA and RNA aptamers and (ii) can be tuned based on the thermodynamics

This journal is©The Royal Society of Chemistry 2019 Chem. Soc. Rev.

Cite this:DOI: 10.1039/c8cs00880a

Duplexed aptamers: history, design, theory, andapplication to biosensing†

Jeffrey D. Munzar, ab Andy Ng ab and David Juncker *abc

Nucleic acid aptamers are single stranded DNA or RNA sequences that specifically bind a cognate ligand.

In addition to their widespread use as stand-alone affinity binding reagents in analytical chemistry,

aptamers have been engineered into a variety of ligand-specific biosensors, termed aptasensors. One of

the most common aptasensor formats is the duplexed aptamer (DA). As defined herein, DAs are

aptasensors containing two nucleic acid elements coupled via Watson–Crick base pairing: (i) an aptamer

sequence, which serves as a ligand-specific receptor, and (ii) an aptamer-complementary element (ACE),

such as a short DNA oligonucleotide, which is designed to hybridize to the aptamer. The ACE competes with

ligand binding, such that DAs generate a signal upon ligand-dependent ACE–aptamer dehybridization. DAs

possess intrinsic advantages over other aptasensor designs. For example, DA biosensing designs generalize

across DNA and RNA aptamers, DAs are compatible with many readout methods, and DAs are inherently

tunable on the basis of nucleic acid hybridization. However, despite their utility and popularity, DAs have

not been well defined in the literature, leading to confusion over the differences between DAs and other

aptasensor formats. In this review, we introduce a framework for DAs based on ACEs, and use this frame-

work to distinguish DAs from other aptasensor formats and to categorize cis- and trans-DA designs. We

then explore the ligand binding dynamics and chemical properties that underpin DA systems, which fall

under conformational selection and induced fit models, and which mirror classical SN1 and SN2 models of

nucleophilic substitution reactions. We further review a variety of in vitro and in vivo applications of DAs

in the chemical and biological sciences, including riboswitches and riboregulators. Finally, we present

future directions of DAs as ligand-responsive nucleic acids. Owing to their tractability, versatility and ease

of engineering, DA biosensors bear a great potential for the development of new applications and

technologies in fields ranging from analytical chemistry and mechanistic modeling to medicine and

synthetic biology.

1. Introduction

Nucleic acid aptamers are single-stranded nucleic acids (mostoften DNA or RNA) that have been selected to bind a definedligand, such as a small molecule or protein, with high affinityand specificity. Aptamers were first discovered in 1990 througha repetitive process of in vitro selection1–3 termed the SystematicEvolution of Ligands by EXponential enrichment (SELEX).2

Roughly a decade after the development of SELEX, naturallyoccurring RNA aptamers were also discovered in the mRNA ofbacteria.4 Given their switch-like gene-regulating behavior,

these aptamer-containing mRNAs were termed riboswitches.Both natural and in vitro-selected aptamers bind their cognateligand with a high affinity (typically with pM to mM affinities).Likewise, aptamer–ligand binding can be highly specific, distin-guishing between closely related molecules by up to 10 000-fold.5

The high affinity and specificity of aptamers lies in their highlyorganized ligand-binding pockets and ligand-stabilized secondarystructures, features that permit multiple non-covalent interactionsto be made between an aptamer and ligand, and that preclude off-target recognition due to steric or ionic factors.6,7

Over the past 25 years, aptamers have emerged as high qualityligand binders, driven, in part, by an ever-increasing repertoire ofavailable aptamers that target an ever-broader diversity ofligands.8–10 Their nucleic acid basis also affords aptamers ever-decreasing production costs via standard synthesis techniques,11

as well as perpetuity and renewability based on the sharing ofprimary nucleic acid sequences. For example, aptamers havebeen implemented as ligand binders in the detection step ofdiagnostic assays, in analytical methods, such as ELISA and

a McGill University and Genome Quebec Innovation Centre, Montreal, Quebec,

Canada. E-mail: [email protected]; Fax: +1-514-398-1790;

Tel: +1-514-398-7676b Department of Biomedical Engineering, McGill University, Montreal, Quebec,

Canadac Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec,

Canada

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cs00880a

Received 7th November 2018

DOI: 10.1039/c8cs00880a

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western blotting,12 for affinity chromatography,13 in therapeuticapplications,14–16 and in nanoparticle-based medicine.17 Today,aptamers and other recombinant binder scaffolds (e.g. antibodyfragments and commercially available modified aptamers, termedSOMAmers) complement traditional antibodies as affinity bindingreagents throughout the chemical and biomedical sciences.12,18

In addition to their popularity as stand-alone ligand bindingreagents, aptamers have been engineered into a variety of aptamer-based biosensors, termed ‘‘aptasensors’’, which are capable ofsimultaneously binding to, and signaling the concentration of,specific ligands of interest. Aptasensors can be designed tointegrate with a variety of readout methods, as summarized in anumber of reviews covering electrochemical aptasensors,19–21

fluorescent aptasensors,22 label-free aptasensors,23 and aptasensorsdesigned to interface with nucleic acid signaling cascades24 orspecifically to sense targets such as pathogens25 or small-molecule ligands.26,27 An exhaustive review of functional apta-sensors, covering many strategies applied in synthetic biology,is also available.28

As detailed in the following section of this review, to transducea signal, aptasensing strategies rely on either (i) modifications toan aptamer’s primary nucleic acid sequence, or (ii) the tendency ofnative aptamers to undergo a ligand-induced structural change6,7

upon binding.29,30 Aptasensors can also be engineered by(iii) interfacing an aptamer with a complementary strand ofDNA or RNA via hybridization, a signaling strategy that isguided by pre-programmed Watson–Crick base pairing.Although popular, this hybridization-based aptasensing strategyhas not been articulated as a separate concept to date, having beenreferred to in the literature using terms such as ‘‘structure-switching aptamers’’,31–33 ‘‘target-induced strand displacement’’aptasensors,34 ‘‘target-induced strand release’’ aptasensors,35 and‘‘switch probes’’,36 among others.

Here, we have chosen to define the duplexed aptamer (DA) asany aptasensor containing two functional nucleic acid elements:(i) a consensus aptamer sequence, which acts as a ligand-specific receptor, and (ii) an aptamer-complementary element(ACE), such as a short DNA oligonucleotide, which is designedto hybridize to a specific portion of the consensus aptamersequence. In DAs, the ACE competes with the ligand for bindingto the aptamer, under the principle that the ligand-dependentdehybridization of the ACE–aptamer duplex can be transducedinto a detectable signal. ACEs can be of different length andcomplementary to different positions of the aptamer, thusimparting DAs with straightforward and programmable designfeatures. In comparison with other aptasensor formats, DA designs

Andy Ng

Andy Ng obtained his PhD in Bio-chemistry from McGill Universitywhere he worked on proteinselection, engineering and structure–activity relationships. Subsequently,he moved to the Institut National dela Recherche Scientifique and devel-oped protein- and aptamer-basedbiosensors. Andy is currently asenior research associate in DrJuncker’s group, with researchinterests in biomolecularengineering and assay develop-ment for environmental monitoring,pathogen detection and diseasediagnosis.

David Juncker

David Juncker conducted his PhDresearch at the IBM ZurichResearch Laboratory from 1999–2002. He then pursued his studiesas a postdoctoral fellow first atIBM Zurich until 2004, and thenone year at the Swiss FederalInstitute of Technology in Zurich.David joined McGill University in2005 as an Assistant Professor inthe Biomedical EngineeringDepartment. He was promoted toAssociate Professor with tenure in2011 and became a Full Professor

in 2016. He currently serves as Chair of the department. David’scurrent interests are in the development and use of micro andnanotechnologies for the generation of improved affinity binders,and for the analysis of proteins, nucleic acids and exosomes in liquidbiopsies and single cells, microfluidic circuits for point-of-carediagnostics, as well as 3D printed and miniaturized organs-on-a-chip.

Jeffrey D. Munzar

Jeffrey Munzar completed hisBEng (Honours) in AeronauticalEngineering in 2011 and his MEngin Combustion in 2013. Heobtained his PhD in Biological andBiomedical Engineering fromMcGill University in 2017, workingunder the supervision of Prof. DavidJuncker. In 2014 he completed ahalf-year internship at the GermanCancer Research Center under thesupervision of Prof. Jorg Hoheisel.After his PhD, Jeffrey carried outpost-doctoral research at McGill

University working to commercialize nanotechnology platforms forblood-based proteomics and the detection of single exosomes. Jeffrey’sinterests include in vitro selection and directed evolution, DNA nano-technology and biosensors, artificial intelligence, and entrepreneurship.

Review Article Chem Soc Rev

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(i) generalize across DNA and RNA aptamers and (ii) can be tunedbased on the thermodynamics of nucleic acid hybridization.Furthermore, DA designs (iii) can be readily integrated with awide range of readout methods, including FRET, fluorescence,electrochemistry and colorimetry (Fig. 1). These advantageshave led to widespread adoption of DAs in biosensing andchemical biology.

In this review, we first (i) provide a history of the develop-ments that inspired the first generation of DA biosensors and(ii) establish a clear distinction between DA and non-DAaptasensing strategies. We then (iii) develop a framework ofDA designs that distinguishes between cis–trans-DA formats,and (iv) discuss SELEX methods that can be used to select forDAs in vitro. Under this framework, we (v) summarize existingtheoretical models of DA ligand binding and (vi) highlightdesign parameters that can be used to tune DA biosensors.We then (vii) describe a number of in vitro and in vivo applicationsof DA biosensors, as well as (viii) gene-regulating applications ofriboswitches and synthetic riboregulators within the context of DAsystems. Finally, we (ix) provide an outlook on future directions ofDAs in the chemical and biomedical sciences.

2. History of aptasensors andduplexed aptamers

Starting in the late 1990’s, aptamers began to be developed intostand-alone signaling biosensors, termed aptasensors. To classifyas an aptasensor, an aptamer must be modified so as to be capableof simultaneously (i) binding the ligand, and (ii) signaling the ligandbinding event. As briefly described below, aptasensors can beengineered to meet these primary requirements by following anumber of strategies. To ensure widespread applicability, however,aptasensor designs should also address secondary practicalrequirements relevant to biosensing, including: (i) the strategymust be applicable to the design of DAs from DNA and RNAaptamers that target a wide range of ligands (generalizability),(ii) the design should require minimal knowledge of the secondaryor tertiary structure of the aptamer–ligand complex (simplicity),(iii) the aptasensor should maintain a high sensitivity for theligand, as close as possible to the affinity of the native aptamer(affinity), (iv) the aptasensor should discriminate the targetligand from structurally dissimilar ligands (specificity), (v) theaptasensor should achieve high signal gain and low background

Fig. 1 Overview of different DA biosensing strategies. Center tile: Schematic diagram of a canonical DA sensing a cognate ligand. Ligand binding resultsin the dehybridization of the ACE–aptamer duplex, and the free ACE (or aptamer) can generate a signal compatible with a range of readout methods.(a) Solution FRET biosensing of ATP. (b) Surface FRET biosensing of ATP and thrombin. (c) Solution fluorescence biosensing of ATP. (d) Surfacefluorescence biosensing of ethanolamine. (e) Surface electrochemical biosensing of thrombin. (f) Surface plasmon resonance spectroscopic biosensingof adenosine. (g) FRET biosensing of ATP using DAs on nanoparticles. (h) Colorimetric biosensing of ATP or cocaine. (i) DA biosensing using nucleic acidcircuits for the detection of cocaine. (j) A synthetic gene network that regulates transcription based on a theophylline DA. (a) is reproduced from ref. 32with permission from the American Chemical Society, copyright 2003. (b) is reproduced from ref. 37 with permission from the American ChemicalSociety, copyright 2011. (c) is reproduced from ref. 38 with permission from the American Chemical Society, copyright 2011. (d) is reproduced fromref. 39 with permission from the American Chemical Society, copyright 2015. (e) is reproduced from ref. 34 with permission from the American ChemicalSociety, copyright 2005. (f) is reproduced from ref. 40 with permission from Elsevier, copyright 2009. (g) is reproduced from ref. 41 with permission fromthe American Chemical Society, copyright 2009. (h) is reproduced from ref. 42 with permission from John Wiley and Sons, copyright 2006. (i) isreproduced from ref. 43 with permission from the American Chemical Society, copyright 2007. (j) is reproduced from ref. 44 with permission fromOxford University Press, copyright 2013.

Chem Soc Rev Review Article

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signal (gain), (vi) the aptasensor should respond quickly to thepresence of ligand (speed), (vii) the performance of the apta-sensor should be easily tunable (tunability), (viii) the aptasensorshould be cost-effective to synthesize (cost), (ix) the designshould be compatible with a range of readout methods (versatility),and (x) the aptasensing strategy should be straightforward toimplement in an assay using commonly available materials,reagents and equipment (practicality).

The first aptasensor design demonstrated in the literaturewas the signaling aptamer (Fig. 2a), created by Ellington andcolleagues by internally labeling an aptamer sequence with afluorescent dye moiety.45,46 To avoid disrupting aptamer functionwhen applying the signaling aptamer strategy, nucleotides near(but not within) the ligand-binding pocket of the aptamer werelabeled, such that ligand binding-dependent changes in thestructure of the aptamer binding pocket resulted in increases(or decreases) in dye fluorescence. Although signaling aptamersoften exhibit affinities close to that of the native aptamer, thisstrategy suffers from numerous limitations. For example, thesignal gain from this design is often low, and the generalizationof this design across aptamers is limited, as the design requiresdetailed knowledge of the native aptamer structure to engineeran aptasensor with a significant fluorescence response to ligandbinding. To address these practical drawbacks when designing newsignaling aptamers, many candidate constructs can be synthesizedwith labels at different bases and tested for optimized signaling,

however this approach adds significant costs and complexity to thedesign process.

In an effort to engineer aptasensors with increased signalgain, aptasensor designs with FRET pairs, rather than singledye moieties, were developed. FRET aptasensor designs wereinspired by molecular beacons47 – DNA hairpins developed tobind complementary DNA or RNA sequences and signal theirconcentrations. Molecular beacons make use of a FRET pair (orfluorescence–quencher pair) of molecular dyes on the beaconconstruct that change in proximity when the beacon opensupon binding a target oligonucleotide, which leads to a largechange in fluorescence. A molecular beacon-like format wasfirst implemented in a split aptasensor by Stojanovic et al., with adesign that is based on splitting a consensus aptamer sequenceinto two subunits, and labeling each subunit with FRET (ordonor/quencher) moieties (Fig. 2b).48 In split aptasensors,ligand-dependent signaling is a function of the proximity of thetwo aptamer subunits: without ligand, the subunits are designedto be unstable, and therefore do not interact on a long timescale;in the presence of ligand, the two subunits assemble to form astable aptamer–ligand complex, in which the FRET pair remainsin close proximity.

A similar FRET signaling strategy also forms the basis fordouble end-labeled aptasensors (Fig. 2c).49–52 The double end-labeled aptasensor design exploits the tendency of a terminalstem (or other double-stranded region) in an aptamer’s native

Fig. 2 Comparison of aptasensing strategies. (a) Signaling aptamer in which the native aptamer is engineered to contain a fluorescent-responsivemoiety located in close proximity to the ligand-binding pocket. (b) Split aptasensor in which an aptamer is divided into two thermodynamically unstablesequences that reassemble only under the presence of ligand. (c) Double end-labeled aptasensor that is engineered to contain a terminal stem that isstabilized by the bound ligand. (d) Overview of a FRET DA biosensor. The DA is engineered by hybridizing a 50 fluorescently labeled aptamer with a 30

quencher labeled ACE. Upon addition of ligand, the ACE is dehybridized from the aptamer, leading to a ligand-dependent increase in fluorescence.


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secondary structure to switch between non-hybridized andhybridized states upon ligand binding. By labeling the ends of thisstem with a FRET or fluorophore–quencher pair, a fluorescencesignal can be obtained as a direct function of the ligand-inducedclosing of the aptamer stem.

Interestingly, molecular beacon-like aptasensor designs allowfor some degree of tunability, either by modifying the degree ofsubunit-to-subunit complementarity for split aptasensors, or bychanging the number of base pairs added (or removed) from theterminal stem for double end-labeled aptasensors. However,the use of molecular beacon-like aptasensors has been largelyconstrained in practice, as the design is not generalizable acrossaptamers. For example, many aptamers do not form a definedstem structure between 50 and 30 ends that can be end labeled.Likewise, most aptamers do not have binding pockets or secondarystructures that can be split into two subunits in a straightforwardmanner (i.e. using a priori knowledge of the aptamer structure)without impacting aptamer activity.

Inspired by advances in molecular beacons and signalingaptamers, the first DA-based aptasensor was developed byHamaguchi et al. in 2000, who employed the canonical thrombinDNA aptamer.53 The aptamer sequence was extended at the 50

end with a short sequence complementary to the 30 end of theaptamer (the ACE), such that in the absence of ligand, the aptamerwas forced to adopt a ‘‘closed’’, hybridized conformation. In thepresence of ligand, the ACE is dehybridized and the aptamer canfreely bind the ligand. Signaling in this DA design relied on thedifferential distance between a fluorophore–quencher pairpositioned on the 50 and 30 ends of the DA sequence.

Subsequently, in 2003 Nutiu and Li engineered a DA bio-sensor using a similar ‘‘duplex-to-complex’’32 strategy, but basedon a bimolecular design (Fig. 2d). Importantly, this DA formatdoes not require any modifications to the native aptamer sequenceor structure, and the ACE can be designed in a straightforwardmanner to hybridize to a defined portion of the native aptamersequence. Indeed, the simplicity in designing and implementingACE–aptamer pairs as DA biosensors forms the most generalizableapproach to aptasensor design. For example, to create a FRETDA, an aptamer can be labeled at the 50 end with a fluorescentmoiety, and a 30 quencher-labeled ACE can be hybridized to the50 end of the aptamer. The ACE serves to simultaneously quenchfluorescence and block a portion of the aptamer consensus

sequence via hybridization. Prior to ligand addition, the DAexhibits low fluorescence, as the fluorophore–quencher pair isin close proximity. Upon ligand addition, the ligand competeswith the ACE to bind to the aptamer, and the ligand-dependentdissociation of the ACE results in a high fluorescence signal. Asa demonstration of the universality of the DA design, Nutiu andLi tested multiple configurations of DAs using ATP- andthrombin-binding aptamers.32 All engineered DAs displayedligand-specific responses with high signal gain, and the respon-siveness of DAs to different temperature ranges could be tunedby adjusting the length of the ACE. Later, Li and colleaguesdemonstrated the same principle for generating DAs based onRNA aptamers,54 further supporting DAs as a generalizableaptasensing design.

As documented in this review, following the first examplesof DAs, additional DA biosensors were rapidly developed forassays targeting a variety of ligands using various readoutmethods. Importantly, all DA designs are guided by two commonprinciples: (i) the ACE is designed to hybridize to a specific portionof the consensus aptamer sequence, and (ii) the generation of asignal by the DA construct ultimately depends on the ligand-dependent dehybridization and/or partitioning of the ACE fromthe aptamer sequence (or inversely, the ligand-dependentdehybridization of the aptamer sequence from the ACE).

3. Duplexed aptamer designs

DA designs can be broadly categorized into those in which (i) theACE and aptamer are separate nucleic acid sequences that arehybridized together, termed trans-DAs (Fig. 3a), or (ii) the ACEand aptamer sequences are contained within the same nucleicacid sequence but are separated by a variable length linker,termed cis-DAs (Fig. 3b). Generally, trans-DAs have lower costsand are more straightforward to design and implement, andthese advantages have led trans-DAs designs to see widespreadadoption in biosensing and in vitro applications (see Sections5.2–5.4 for detailed examples). Conversely, cis-DAs, which sharedesign similarities with natural riboswitches, are more prevalentin applications where the aptamer and ACE must remain in closeproximity or be present on the same nucleic acid construct, forexample in real-time, reusable and/or therapeutic applications,

Fig. 3 Comparison of cis- and trans-DA designs. (a) Overview of trans-DAs, in which the ACE and aptamer sequences are separate nucleic acids. Uponligand binding, the ACE is free to dissociate into solution. (b) Overview of cis-DAs, in which the ACE is incorporated within the same nucleic acid strand asthe aptamer, but is separated from the aptamer by a variable length linker. Upon ligand binding, the ACE dehybridizes from the aptamer but remains inclose proximity to the aptamer–ligand complex.


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and in synthetic biology (see Sections 5.5 and 5.8 for detailedexamples).

In this section, we introduce general design concepts anddevelop a framework for DA biosensors. We describe differentfactors related to DA labeling and ACE design that should beconsidered when engineering cis- and trans-DAs from existingaptamers, giving some examples where necessary. We alsoconsider and discuss special cases of cis- and trans-DA designs,and contrast these cases with native aptamers. Finally, weexplore SELEX strategies that can be used to artificially generatetrans-DAs in vitro.

3.1 trans-DAs

3.1.1 Labeling. The first DA biosensors were engineered bysynthesizing the ACE as a separate oligonucleotide sequencefrom the aptamer, and this approach forms the basis for trans-DA designs. For most trans-DA biosensors, a fluorescent, elec-trochemical or other type of label is used to generate a signal,and this label can be incorporated into the aptamer or the ACEsequence in a variety of manners, so long as the assay format ofinterest is taken into account. For example, DA labeling requiresdifferent considerations for DA deployment in surface vs. solutionassays, in assays using time course vs. end-point measurements, orin assays using equilibrium vs. non-equilibrium readouts. As ageneral design rule, for aptamers with known structures, labelingof bases in the aptamer known to be actively involved in ligandrecognition and/or formation of the ligand-binding pocket shouldbe avoided, as labeling of these bases could interfere with aptamerfunction. For aptamers with unknown ligand-bound structures, anexcellent strategy is to terminally label the aptamer, or toincorporate short 50 or 30 extensions to the aptamer consensussequence and append these with a signaling moiety. Shortextensions to the aptamer can also be used as toeholds forACE hybridization, allowing DAs to be engineered with ACEsthat are partially hybridized to the aptamer consensus sequenceand partially hybridized to the extension toehold.

For single-labeled, surface DA biosensors, such as fluorescenceDAs and most electrochemical DAs, either the aptamer or the ACEcan be labeled, so long as it is possible to partition releasedaptamer (or ACE) molecules from the surface in order to measurea signal. For example, a surface DA can be assembled using labeledaptamers hybridized to surface-conjugated ACEs, or inversely,using labeled ACEs hybridized to surface-conjugated aptamers;in both designs, signal generation ultimately depends on thereleased labeled strand being partitioned from the surface-anchored strand (Fig. 4a). Single-labeled, surface DA systems canalso be inverted by using a label on the surface-immobilized DAelement, so long as the release of the second DA element changesthe signal of the label on the surface-tethered element. It isimportant to note that solid surfaces can have a large impact onhybridization and biosensing55–58 and must be taken intoaccount when implementing surface DAs. As a rule of thumb,a minimum spacer length of 10–20 nucleotides between theaptamer (or ACE) and the surface should be added to minimizesteric impacts of the surface on aptamer–ligand recognition oraptamer–ACE hybridization.59,60

In contrast, dual-labeled trans-DAs, such as FRET DAs, relyon a ligand-dependent increase (or decrease) in fluorescencesignal that is a function of the distance between the two labels.Many strategies can be used to engineer FRET DAs (Fig. 4b).For surface and solution assays, the aptamer is most oftenlabeled with one FRET moiety, while the ACE is labeled with amatching moiety, such that the hybridized aptamer–ACE duplexpositions the FRET pair in close proximity. FRET signaling canalso be accomplished using a single fluorophore-labeled DA incombination with gold nanoparticles (AuNPs), which serve bothas the surface for DA immobilization and as the quencher(Fig. 4b).

Fig. 4 Design configurations of trans-DAs. Candidate labeling sites forsurface DA designs are shown as red hexagons. (a) Overview of strategiesto separate ACE and aptamer sequences for DAs in surface assays. Eitherthe aptamer or the ACE can be immobilized on the surface. (b) Overview ofFRET DAs designed for solution (left), surface (middle) and AuNP (right)assays. Only the initial DA structure is shown here; ligand binding wouldproceed as in (a). (c) Example of a tripartite FRET DA biosensor, in which a thirdstrand that caries a fluorophore (in pink) is hybridized to a long single-stranded50 aptamer extension (in purple). Upon ligand binding, the quencher-labeledACE dehybridizes from the aptamer sequence, whereas the fluorophore-containing third strand remains hybridized to the extension and generates anincreased fluorescence signal. (d) Example of an ACE with a pre-programmedhairpin structure used to engineer improved electrochemical DAs (left). TheACE self-hybridizes upon aptamer dissociation, generating a higher signal gainvia an increase in the electron transfer (eT) rate. Neutralizer ACEs incorporatingpositively charged moieties can also be used to neutralize the negativecharges present on the sugar phosphate backbone of an aptamer, generatingDA biosensors with improved eT rates before ligand addition (right).


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Rather than implementing labels on both the aptamer andthe ACE, trans-DAs can also be designed as tripartite nucleicacid systems, with a third nucleic acid sequence carrying a labelinvolved in signal generation or readout (Fig. 4c). For example,building on the traditional bipartite DA design, Nutiu and Liimplemented an ATP aptamer sequence that was appendedwith a long single-stranded 50 extension. To form a DA, theaptamer was then hybridized with (i) a quencher-labeled ACE, aswell as (ii) a fluorophore-carrying oligonucleotide that hybridizedonly to the 50 aptamer extension.32 In this tripartite DA design,following ligand binding and ACE dehybridization, the fluorophore-labeled signaling strand remains hybridized to the 50 aptamerextension and produces a strong fluorescent signal. This tripartitestrategy can also be used to design DAs that are resistant tofalse-positive readouts due to enzymatic degradation of theaptamer or ACE nucleic acid sequences, for example by usingreporter oligonucleotides that bind to the 50 and 30 ends of anextended aptamer.61

3.1.2 ACE design. Another modifiable design element fortrans-DAs is the structure of the duplexing ACE. For example,double-end-labeled ACEs can be engineered to form a hairpinwhen dehybridized from the aptamer, which allows for bothFRET labels to be transferred onto an ACE, as demonstrated byNi and Ho,62 or to generate electrochemical DAs with increasedsignal gain, as done by Mao and colleagues (Fig. 4d).63 Transferringthe entire reporting system to the ACE can also be used to integrateDAs with other readout mechanisms, such as nanoparticle colori-metric systems and nucleic acid circuits (see Section 5 for examples).ACEs can also be modified with additional moieties for biosensingpurposes. For example, in electrochemical applications, neutralizerACEs can be designed to contain positively-charged moieties,which reduce the negative charges present on the sensor surfaceand has been shown to improve DA biosensor performance byincreasing signal gain (Fig. 4d).64 ACEs can also be designed withintentional base pair mismatches, which can be used to eitherdestabilize the ACE–aptamer duplex, or to permit even longerACE sequences to be used. The effect of base mismatches shouldalso be considered if aptamers with modified nucleosides, suchas SOMAmers, are implemented – ACEs may not form canonicalduplexes, leading to weaker ACE–aptamer duplexes than in non-modified aptamers.

3.2 cis-DAs

For some applications, a drawback to using trans-DAs is theneed for two separate aptamer and ACE elements in the DAdesign. This is particularly problematic for continuous and/orlong-term biosensing, since the loss of any DA elements leadsto degrading sensor performance over time. The drawback canbe overcome by incorporating the aptamer and ACE within thesame nucleic acid sequence to form a cis-DA with a variablelength linker (Fig. 5a). The first cis-DA was engineered with azero-length linker (and originally termed an ‘‘aptamer beacon’’)by Hamaguchi, Ellington and Stanton.53 Subsequently, a cis-DAbased on a FRET pair was implemented using a PEG linkerbetween the aptamer and the ACE.36 This design has also beenreferred to as a ‘‘switch probe’’ by Tan and colleagues, owing to theability of cis-DAs to rapidly interconvert between ACE-duplexed andACE-free states.36 Although we make a distinction here between cis-and trans-DAs, both designs share similarly tunable parameters,including label selection and label positioning.

3.2.1 Labeling. cis-DAs have a number of design optionsfor labeling. For cis-DAs operated in solution assays, for examplewith FRET DAs, the FRET pair can be located on the samenucleic acid sequence as the aptamer and ACE, most often onthe extreme 50 and 30 ends of the DA, or extensions to theaptamer sequence can be labeled (Fig. 5a). Alternatively, anothernucleic acid can be added to the system to carry one of thelabels, akin to tripartite trans-DAs described in Section 3.1.

For surface-immobilized cis-DAs in which the surface isactively involved in signaling, for example in electrochemicaland gold nanoparticle FRET systems, DAs can be implementedwith a single redox or fluorescent label, as the surface acts asthe second label (Fig. 5b). cis-DAs can also be implemented aslabel-free DAs when using appropriate readout methods, such assensitive SPR systems, or in electrochemical systems incorporatinga free redox reagent (such as Fe(CN)6

3�) in the buffer that isdifferentially repelled by the ligand-bound and ligand-free cis-DA(see Section 5 for examples).

3.2.2 ACE design. A key difference between cis- and trans-DA designs is the minimum length of the ACE that can be used toengineer a DA. Importantly, for the same length ACE, cis-DAs areexpected to have higher ACE–aptamer hybridization affinitiesand faster ACE–aptamer hybridization on-rates, owing to the

Fig. 5 Overview of design strategies for cis-DAs. Candidate labeling sites for cis-DA designs are shown as red hexagons. (a) cis-DAs can be implementedwith a zero length (left) or a variable length linker (right) between the ACE and the consensus aptamer sequence. (b) Example of a single-labeled DA forelectrochemical applications, shown here with the aptamer immobilized nearest to the surface. Upon ligand binding and ACE dehybridization, theelectrochemical label on the dehybridized ACE is located further from the EC surface, decreasing the eT rate.


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increased effective local concentration of cis-located elements.As a result, ACEs in cis-DAs more easily compete with the ligandfor binding to the aptamer, and shorter ACEs can therefore beimplemented in cis- vs. trans-DAs. For example, 7-mer ACEshave been used for engineering cis-DAs for ATP,36 whereas 9- to12-mer ACEs were suitable for engineering trans-DAs for ATP32

(see Section 4 for details and additional discussion on themodeling of ACEs as part of the ligand binding dynamics ofDAs, and the impact of different ACE lengths and locations onDA biosensors).

3.2.3 Comparison of trans-DAs, cis-DAs and native aptamers.cis-DAs are in some ways similar to native aptamers. In cis-DAs,the ACE forms a stem-loop structure when hybridized with theaptamer, a configuration that is similar to the intrinsic secondarystructure of many native aptamers, which often contain internalstem, stem-loop or pseudoknot elements. Although DAs sharesecondary structures in common with aptamers, native aptamersmake use of these structures directly for ligand interaction,and in this sense the removal or perturbation of an aptamer’ssecondary structure will often result in loss of function. Incontrast, the structure of an ACE-duplexed aptamer is notoptimized for ligand binding, as the ACE competes with ligandbinding; rather, the ACE-free native aptamer is the most favor-able structure for ligand binding.

Whereas cis-DAs are typically implemented with short linkersseparating the ACE and aptamer, it is interesting to consider theextreme case of a cis-DA with a very long linker between theaptamer and the ACE. Such a cis-DA would likely behave asthough the ACE and aptamer were separate elements. As such, atrans-DA might be considered a special case of a cis-DA in whichthe linker length is very long; for a solution assay, this distancewould be on the order of the mean free path length of dehybridizedaptamer and ACE molecules. Similarly, we note that a trans-DA inwhich the consensus aptamer sequence has been appended with a

very long single-stranded toehold sequence designed for ACEhybridization might never undergo complete dehybridization,even after aptamer–ligand binding and dehybridization of theportion of the ACE that duplexes the aptamer sequence. Such atrans-DA might therefore be better classified as a special case ofa zero-length cis-DA, as the ACE is expected to remain in closeproximity to the aptamer after ligand binding, owing to theremaining ACE-toehold duplex.

3.3 DA SELEX

Starting from an initial pool of nucleic acids sequences, SELEXamplifies those that are most fit for the selection pressuresimplemented. First-generation SELEX methods focused on thediscovery of high affinity, high specificity aptamers by imposingstrict partitioning constraints. However, SELEX can also be modifiedto generate aptamers with other functions, such as intrinsicfluorescence signaling,46 by imposing different selection constraints.Commonly implemented modifications to traditional SELEX,and recent advancements in the field, have been reviewedelesewhere.65–67

Interestingly, SELEX can also be adapted for the directselection of DAs, as shown by Nutiu and Li in 2005 (Fig. 6).68

DA SELEX includes four key modifications to standard SELEX:(i) incorporation of a defined region in the aptamer library forACE hybridization, (ii) inclusion of an ACE-library immobiliza-tion step that enforces an ACE hybridization selection pressure,(iii) rejection of library members that spontaneously dissociatefrom the ACE, thereby selecting for stable DAs under buffer-onlyconditions, and (iv) selection of aptamers that dehybridize rapidlyfrom the ACE when incubated with a ligand, enriching for themost ligand responsive DAs. Akin to traditional SELEX, multiplerounds of library immobilization via hybridization, ligand-induceddehybridization, partitioning and library amplification are carriedout in DA SELEX to generate ligand-specific DAs.

Fig. 6 In vitro selection of ligand-responsive DAs. The aptamer library was designed with two random regions (N10 and N30 domains) separated by aconstant 15-mer domain. A 15-mer ACE, complementary to the 15-mer domain, was immobilized on beads and used to pull-down the library viahybridization. Upon addition of ligand, sequences within the library that bind the ligand and that were released from the ACE were eluted into solution.The eluted pool was amplified, and the SELEX process was repeated for 18 rounds, resulting in the selection of DAs specific for ATP and GTP. Reproducedfrom ref. 68 with permission from John Wiley and Sons, copyright 2005.


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In the first implementation of a DA SELEX experiment,aptamers against ATP and GTP as ligands were selected usinga defined 15-mer ACE for library pull-down.68 Interestingly, allaptamer sequences recovered after 18 rounds of panning hadeither a single base mutation or deletion within the 15-merconstant region of the aptamer designed for ACE hybridization.These mutations arose from errors in library synthesis or libraryamplification that were enriched during subsequent panningrounds. A single mismatched base pair leads to significantlyweaker hybridization between ACEs and the aptamer,69 andwhile a mismatched aptamer–ACE duplex is expected to create aDA that is more susceptible to dissociation under buffer-onlyconditions, mismatches presumably arose during DA SELEXdue to the strong selection pressure for DAs with weaker ACE–aptamer duplexes that more favorably switch structures in thepresence of ligand. This finding supports DA ligand bindingsensitivity being a function of ACE hybridization affinity, andthis thermodynamic aspect of DA ligand binding is discussed inmore detail in Section 4. However, the additional requirementfor low DA dissociation rates under buffer-only incubationensured that any ACE–aptamer duplexes that were too weaklybound, for example any aptamer sequences with multiple mis-matches to the ACE, were depleted during SELEX.

SELEX has also been used to generate DAs against theantibiotic kanamycin A.70,71 Coined ‘‘capture-SELEX’’ by Strehlitzet al., this method made use of a shorter 12-mer ACE (anda matching defined domain in the aptamer library) forimmobilization. After 13 rounds of selection, enriched aptamersequences also contained base pair mismatches or deletionswith the 12-mer ACE, further demonstrating the tendency ofACE hybridization affinity to respond to DA SELEX selectionpressures. SELEX has also been used to generate DAs againstmelamine,72 and a version of DA SELEX that makes use of one ofthe primer regions in the aptamer library for immobilization hasbeen implemented for the generation of DAs that target zinc.73

We note that other SELEX methods have been demonstratedthat make use of oligonucleotide hybridization for libraryimmobilization and ligand-induced release.74–76 Although thesemethods generate aptasensors, they do not classify as DAs as theydo not contain ACEs. Rather, these aptasensors make use of apre-programmed strand-displacement circuit that is activatedwhen the aptamer binds a ligand, akin to double end-labeledaptasensors. Specifically, for some library members, the splitsegments of the circuit are brought into close proximity uponaptamer–ligand binding, leading to the DNA strand displacement-mediated release of these sequences into solution.

4. Tuning and modeling of duplexedaptamer ligand binding

A key factor driving the adoption of DA biosensors is thecapability to design DAs with predictable and tunable performance.A large importance must therefore be placed on understanding themechanisms and variables that underlie the performance ofDAs. In biosensing applications, researchers often aim to detect

and quantify an analyte known to be present within a limitedconcentration range in a sample of interest. While DA bio-sensors are often engineered with maximized affinities, inpractice, DAs must be engineered with affinities that align witha ligand’s concentration range within the real (or buffer-diluted)sample, as the affinity of the DA directly determines the limit ofdetection and dynamic range of quantification of the biosensor.In this section, we outline theoretical models of DA ligandbinding and binding kinetics, and describe how these modelscan be applied to engineer DAs with desired ligand bindingaffinities.

Key DA biosensor design considerations include (i) whatassay format should be implement for a desired DA application,(ii) what buffers, readout methods or other associated technologiesshould be used to optimize a DA system, (iii) what length ACEshould be used to generate an optimized DA biosensor, and (iv)where in the aptamer sequence should an ACE be hybridized foroptimized biosensor performance. The term optimized is to beunderstood with respect to the intended application of the DA andthe main parameters that govern the utility of generated DAbiosensors – as outlined in Section 2, these include biosensingproperties such as affinity, selectivity, gain, and speed, as well asmore general properties such as generalizability, simplicity,tunability, cost, versatility, and practicality. These factors areco-dependent and therefore most applications of biosensorsrequire trade-offs among these parameters or with other facetsof biosensor design, including sample type and volume, sensortype, assay complexity, portability, and/or end user-friendliness.

We touch on design considerations (iii) and (iv) in detail inSections 4.1–4.5 below. In addressing design consideration (i),DA biosensors can be reliably designed for surface- or solution-based implementation, with both designs being amenable toeither non-equilibrium- (i.e. partitioned or non-homogenous)or equilibrium-based (i.e. homogenous) assay formats. However,the design and assay format of a DA biosensor largely dependson the intended application. In this regard, we review a varietyof DA designs and assay formats in Section 5. Additionally, theligand binding dynamics underlying DA performance differwhen DAs are operated in non-equilibrium vs. equilibrium assayformats, as described in Section 4.2 below.

For design consideration (ii), generally, DAs should be imple-mented in buffers at the same pH and containing similar cations(and ideally similar concentrations of cations) as that used forthe in vitro selection of the native aptamer. This ensures that theaptamer will maintain the capability to recognize and bind theligand of interest with high affinity and specificity. In contrast,the choice of DA design and readout method largely depends onthe intended application. For example, in assays with repeatedreadout steps, it might be optimal to use cis-DAs together withstringent wash steps to refresh the sensing surface betweenbiosensing rounds, rather than rely on the rehybridization ofthe aptamer–ACE duplex after each round when using a trans-DA design. For point of care devices, colorimetric readouts andlateral flow assays allow users to operate DAs without additionalinstruments using naked-eye readout, reducing costs andcomplexity. In contrast, for assays requiring high sensitivity,


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electrochemical DAs or single-molecule readout technologiesmight be best suited, as these readout methods permit lowerbiosensing detection limits.

4.1 Models of ligand binding

Two models are commonly employed to describe ligand-bindersystems, termed conformational selection (MWC model77) andinduced fit (KNF model78) (Fig. 7). In conformational selection,a binder is modeled as existing at equilibrium between bindingcompetent and non-competent states, and the ligand is assumedto only bind the competent state, so as to form a ligand-boundstructure. In contrast, induced fit presumes that the ligand is

capable of catalytically interacting with the non-competentbinder state, directly inducing its fit in the binder and resultingin the formation of a final ligand-bound structure.

Conformational selection and induced fit binding pathwayshave been used to describe a number of protein80–84 and nucleicacid85–87 systems (including native aptamers), as well as complexphenomena observed in nature, including allosteric interactionsand signaling cascades.88,89 Conformational selection and inductedfit are not mutually exclusive, and as shown in Fig. 7, represent twoparallel pathways from an unbound to bound state, or expresseddifferently, represent a 4-state system. However, it is not always clearwhich binding mechanism dominates a dynamic ligand-bindersystem, or if the mechanisms co-exist.79,90–94 For example, whereassome natural riboswitch aptamers have been documented to bindtheir cognate ligand via conformational selection,95,96 others areregulated via an induced fit mechanism or a combination of thetwo mechanisms.93,97,98

4.2 Models of DA–ligand binding

DA–ligand binding has most commonly been described underthe framework of purely conformational selection. A conformationalselection model of ligand binding imposes that a DA does notdirectly sense ligand, and the ACE is required to first dehybridizefrom the aptamer, followed by binding of the natively foldedaptamer and the ligand (pathway including states 1, 2 and 3 inFig. 7 and 8); the dehybridization of the ACE and the aptamer–ligand binding are independent events, implying that the ACEdehybridizes (completely) without influence by the ligand.99

Under a conformational selection model, the introduction ofligand to a DA system stabilizes the non-duplexed aptamer state(which binds the ligand with an affinity of KApt

d ), and thisrepopulation of available aptamer states drives a ligand-dependent signal. Thus, the hybridization affinity of an ACE

Fig. 7 Conformational selection and induced fit are alternative bindingpathways used to describe ligand-binder systems. An overview of athermodynamic cycle for a model protein (in green) binding a ligand (L, inred) is shown, outlining conformational selection and induced fit pathways.Binding non-competent (1), binding competent (2), ligand bound (3) andinduced fit intermediate (4) states are labeled. Reproduced from ref. 79 withpermission from the American Chemical Society, copyright 2013.

Fig. 8 Overview of conformational selection and induced fit models of DA ligand binding. Both candidate pathways are shown for a solution FRET DAcomprising a 32-mer ATP DNA aptamer hybridized by a 12-mer ACE. The predicted affinity of the DA depends on the existence of conformationalselection (KC.S.

d , states 1–2–3) or induced fit (KI.F.d , states 1–10–3) pathways, which are special cases of a general 4 state model of DA ligand-binding (KObs

d ,cycle –1–10–3–2–). Reproduced from ref. 100 with permission from The Royal Society of Chemistry, copyright 2017.


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(KHyb – as a function of ACE length, ACE GC-content, ACEconcentration, assay temperature, and buffer conditions), butnot ACE location, would be expected to modify the affinityof engineered DAs, with DA–ligand affinity always lower thannative aptamer–ligand affinity. For DAs operating with a con-formational selection binding mechanism, the kinetics andaffinities of the system are largely governed by the hybridizationkinetics of the ACE, as in practice it constitutes the limiting step inthe pathway. According to this model, the longer (i.e. more stable)the ACEs, the slower the sensor response and the lower the affinity.

In contrast, an induced fit model of DA ligand bindingconsiders the ACE-duplexed aptamer as capable of directly bindingthe ligand from the ACE-hybridized state. This binding event formsa ternary ACE–aptamer–ligand complex, which is followed bydehybridization of the ACE to yield the final aptamer–ligandcomplex (pathway including states 1, 4 and 3 in Fig. 7, and 1, 10

and 3 in Fig. 8). Induced fit therefore models the ligand as a catalystthat is sensed by the DA with a certain affinity (KFit), and thebinding of the ligand promotes the ensuing dehybridization of theACE from the aptamer (as a function of KHyb*), ultimately leadingto a ligand-dependent signal. Under this model, both the lengthand location of an ACE are tunable parameters, since the responseof a DA to a catalytic ligand may be dependent on both the ligand–aptamer affinity, the ACE hybridization affinity, the 3D structure ofthe DA, and the availability of initial ligand recognition sites in theACE-hybridized aptamer that promote induced fit binding. For DAsoperating with an induced fit binding mechanism, the kinetics andaffinities of the system are therefore highly dependent on ACEposition and length, and the impact on sensor kinetics may beexpected to vary greatly for different aptamers and different ACEs,similar to differences in enzyme behavior and/or protein inter-actions in the presence of cofactors.

However, conformational selection and induced fit bindingmechanisms are not mutually exclusive, and both pathwaysmay be at play to varying degrees in a DA system. In this case ofDAs, conformational selection and induced fit pathways can bemodeled as a 4-state thermodynamic cycle with two bindingpathways that operate at equilibrium (states 1, 2, 4, 3 in Fig. 7,and 1, 10, 2 and 3 in Fig. 8), and the expected affinity of a DAwould be a function of the variables driving both pathways.90,100

Interestingly, we note that the ligand–DA binding mechanismsdescribed above mirror nucleophilic substitution reactions inorganic chemistry,101 in which a nucleophile selectively attacks asubstrate (comprised of a saturated carbon and a leaving group)and replaces the leaving group. When paralleled with DAs, in anucleophilic substitution reaction the substrate represents the DA,the nucleophile takes on the role of the ligand, and the leavinggroup represents the dehybridized ACE. Here, the SN1 modelmirrors the conformational selection pathway in DAs, since theunfavorable de-hybridization of the ACE from the aptamer (a slow,rate limiting step) is a prerequisite to reach an intermediate statethat allows for the binding of the ligand to the aptamer (rapidstep). In contrast, the SN2 model mirrors an induced fit bindingmechanism for DAs, with the ligand directly displacing the ACE.Although the parallels between nucleophilic substitution and DAbinding have not yet been explored in the literature, we note that

the study of DA ligand binding could benefit from the experi-mental and conceptual frameworks developed in the study ofnucleophilic substitution reactions.

An additional important consideration for DA binding dynamicssurrounds the assay format implemented for biosensing. For DAsystems that are operated at equilibrium, for example in homo-geneous cis- or trans-DAs operated in the solution phase (andlikewise for surface-assembled cis-DAs), all intermediate DAstates (1, 10, 2 and 3) co-exist as a function of the affinityconstants of the system and the concentrations of the aptamer,ACE and ligand (Fig. 8). In practice, the observed affinity of a DAsystem is dependent on the existence of each binding mechanism,and accordingly the mathematical derivation of the observedaffinity of the DA biosensor depends on the mechanism at play(in the case where only conformational selection or only inducedfit exists) – in the general case, the observed affinity of a DA atequilibrium is a function of three of the four affinity constants fora 4-state cycle.100,102 Hence, the binding affinity and bindingdynamics of DAs operated at equilibrium are a function of bothconformational selection and induced fit kinetics, and aptamer,ACE and ligand concentrations. Optimized equilibrium DAsshould therefore be designed by considering the kinetics ofboth ligand binding pathways.

DAs can also be implemented with non-equilibrium kineticsby using assay formats in which the release of one DA elementis essentially irreversible, or by tracking the binding kinetics ofthe DA in real time before equilibrium is reached. In this design,induced fit is expected to have a large impact on biosensingperformance. For example, surface trans-DAs can be engineeredin which an ACE is covalently conjugated to the biosensingsurface, the aptamer is hybridized to form a DA, and unhybridizedaptamer molecules are removed prior to biosensing. This sensorsurface can be imaged under conditions that prevent releasedaptamers from rehybridizing, such as in a large sample volumeor by using a microfluidic flow cell to continuously resupplysample solution. For such a non-equilibrium surface DA, assaysignal is generated as a function of (i) induced fit ligand bindingby the surface DA, which ultimately promotes the ligand-dependentdehybridization of the aptamer from the ACE (ligand specific signal,via induced fit pathway 1–10–3) or (ii) spontaneous dissociation ofaptamer molecules from ACEs into the sample volume (backgroundsignal, via conformation selection pathway 1–2). Due to thenegligible rate of rehybridization of free aptamer strands, forsuch non-equilibrium assays, the kinetics of a surface DA can besimplified by considering only states 1 and 10, since DAs instates 2 or 3, once formed, only exist in the sample volume. Suchnon-equilibrium DAs can be optimized by (i) minimizing thespontaneous dissociation rate of pre-assembled DAs (i.e. minimizethe off rate, koff, of aptamer–ACE dehybridization between states 1and 2), (ii) maximizing the induced fit affinity of the DA for theligand (i.e. use ACEs with maximum KFit), and (iii) maximizing theinduced fit dissociation rate of pre-assembled DAs (i.e. maximize theligand-induced off rate, koff*, of aptamer–ACE dehybridizationbetween states 10 and 3). Thus, while the design of optimalnon-equilibrium surface DAs requires optimization of bothpathways, the role of induced fit ligand binding as a catalytic


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signal generation mechanism places a large importance onselecting the optimal ACE for the aptamer when designing anon-equilibrium DA biosensor for an intended application – inparticular, induced fit can lead to faster binding kinetics in DAsystems, and this is explored in more detail below.

4.3 Conformational selection model of DAs

DAs (and other aptasensors) have traditionally been designed,modeled and analyzed using a conformational selection modelof ligand binding.99,103–110 For example, Li and colleaguesproposed a 3-state model of solution FRET DAs that includedthe possibility of misfolded aptamer states, and fit the kineticrates of a DA system to real-time fluorescence measurements.103

Subsequently, building on their work on nucleic acid molecularbeacons,105 Plaxco, Ricci, Vallee-Belisle and colleagues developeda model of cocaine DNA DAs (and cocaine dual end-labeledaptasensors) based on conformational selection, in which ACEsare modeled as allosteric inhibitors, serving to sequester cocaineDNA aptamers into a non-ligand responsive, duplexed state (Fig. 9).99

Conveniently, the validity of a conformational selectionmodel of DA ligand binding would enable researchers toleverage models of nucleic acid hybridization,111,112 as well asonline tools for the prediction of hybridization affinities (e.g.mfold/DINAMelt113), for the rational design of DAs. In principle,this model allows for DA affinity to be tuned based on only(i) the affinity of the native aptamer for the ligand (K Apt

d ) and(ii) the hybridization affinity of the aptamer–ACE duplex (KHyb).For example, Porchetta et al. used such a model to rationallytune the affinity of cocaine DNA DAs using ACEs varying in KHyb

(by modifying ACE length), achieving control over more thanthree orders of magnitude.99 Interestingly, the response kineticsof cocaine DNA DAs with longer ACEs was observed to signifi-cantly decrease, as expected for DAs with a conformationalselection binding mechanism. This model also allows for thedynamic range of DA biosensors to be rationally extended ornarrowed over the typical 81-fold range114 of ligand concentrationsensitivity, either by combining sets of DAs with differing KHyb, orby implementing the DA together with non-signaling aptamermolecules that serve as ligand depletants, respectively.99 Moregenerally, when designing a DA system based on a conformationalselection binding mechanism, affinity isolines can be used toguide the design of trans-DAs as a function of KApt

d , KHyb and ACEconcentration ([ACE]) (Fig. 10a).100 Conformational selection hasbeen applied successfully to model other functional nucleic acidscontaining aptamers, including ribozymes,115 shRNA switches116

and riboswitches117 (the relationship between DAs and ribo-switches is discussed in Section 5.8).

For trans-DAs, under a conformational selection model, themost straightforward parameter to tune is KHyb, based on ACElength and GC-content (i.e. ACE–aptamer DG). Assay conditionscan also be modified to tune ACE hybridization affinities. Forexample, KHyb will increase with increasing concentration ofmonovalent (e.g. Na+ and K+) or divalent (e.g. Mg2+) ions in thebuffer, by operating the assay at a lower temperature, or byimplementing FRET or fluorophore–quencher pairs known tointeract and stabilize nucleic acid duplexes.118,119 KHyb can alsobe increased or decreased by using buffer additives, such asbetaine, DMSO, formamide, urea, or tetramethyl ammoniumchloride (TMAC), which impact A-T and G-C hybridizationstabilities. When tuning trans-DAs, it is helpful to consider a‘‘Rule of 7 of Watson–Crick base pairing’’ in the design of stableduplexes,69 which stipulates that at least 7 consecutive basespairs are needed in the design of stable nucleic acid duplexes,thereby ensuring that ACEs remain stably hybridized to theaptamer under buffer-only conditions. We note that most trans-DAs implemented in the literature indeed use ACEs that fall inthe range of 9- to 15-mers (Fig. 10b). The successful implementationof DAs with ACEs longer than 20-mers is somewhat unexpectedaccording to a conformational selection model of ligand binding.Indeed, for long ACEs, the hybridized state is much favored over thedehybridized state, thus creating a bottleneck in the ligand bindingpathway, shifting equilibrium towards the hybridized state, andresulting in very slow binding kinetics. In contrast, an induced fitmodel affords a straightforward explanation for ligand binding ofDAs with long ACEs, as described in Section 4.4 below.

For cis-DAs, ACE length can be tuned via affinity isolines asdescribed for trans-DAs, albeit predicted DA affinities are nolonger a function of ACE concentration. Rather, the length ofthe spacing loop between that ACE and aptamer can be used totune DA affinities as a function of ACE hybridization kinetics.Although the opening rate of a cis-DA is expected to be relativelyindependent of spacer length, the closing rate is a function ofspacer length (with shorter spacers leading to faster ACEbinding due to a higher local concentration of the interactingpair), as linker length is the primary driver of the probability of

Fig. 9 Conformational selection model of aptasensor ligand binding. (a) Adual end-labeled aptasensor can be modeled as exchanging betweensingle-stranded and hybridized stem states, with a switching affinity Ks,whereas the binding competent state is modeled as sensing the ligand withan intrinsic affinity Kint

d . (b) Similarly, a DA can be modeled as exchangingbetween ACE-hybridized and ACE-free structures with a hybridizationaffinity Ks (i.e. KHyb), with the ACE-free aptamer binding the cognate ligandwith an intrinsic affinity Kint

d (i.e. KAptd ). Reproduced from ref. 99 with

permission from the American Chemical Society, copyright 2012.


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end-to-end collisions between ACE and aptamer DA elements,similar to molecular beacons.120 Given their intrastrand nature,the ACEs in cis-DAs therefore exhibit higher hybridizationaffinities, and cis-DAs can therefore be designed with corre-spondingly shorter ACEs than trans-DAs.36 This aspect of cis-DAdesign is well reflected in the literature, with most cis-DAshaving been designed with 5- to 10-mer ACEs (Fig. 10b).

4.4 Induced fit model of DAs

A number of DA binding observations from the literature arenot readily explained by a conformational selection-only model

of ligand binding, which led us to propose an induced fit modelof DA ligand binding.100,121 We found solution-phase DAsagainst ATP with much higher ligand affinities for ATP thanpredicted by conformational selection, up to one million-foldhigher, Fig. 10a.100 The conformational selection pathway, byits definition, requires first the complete dissociation of theACE from the aptamer, which is followed by the binding of theaptamer to the ligand. Hence it would be expected that DAsengineered with long ACEs (for example, trans-DAs with ACEslonger than 20-mers) would be inefficient for binding theirligand because the de-hybridization is highly unfavorable, andthe binding would be expected to be very slow. This scenarioalso implies a low binding affinity, yet some DAs bind theirligand with high affinity and at relatively high kinetic rates, asdocumented extensively for the ATP DNA aptamer (e.g. ref. 32).Limitations of the conformational selection model, as describedabove, were first reported by Ellington and colleagues, followingthe observation of much-faster-than-expected binding kineticsof a thrombin-binding trans-DA in solution.122

Additionally, as described at the end of Section 4.2, thesignal generation mechanism for non-equilibrium surface DAs(which includes many electrochemical non-equilibrium trans-DAs in the literature) is not readily explained by a conformationalselection-only model of ligand binding. These biosensors oftendeploy an aptamer that is hybridized to a surface-immobilizedACE, and the surface is queried over time for e.g. changes inelectrochemical potential in the presence of ligand. Under amodel of conformational selection, the aptamer is modelled asfirst dissociating from the surface-bound ACE, followed by a bindingevent in solution. However, aptamers that have dehybridized froman ACE are no longer localized to the surface, and hence anyaptamer–ligand binding event should not lead to a signal on thesurface. Hence, from a mechanistic standpoint, it remains unclearhow aptamer–ligand interactions are ultimately transduced into aligand-dependent surface signal for these DAs.

Interestingly, induced fit has been used to describe thebinding dynamics of native aptamers,7,123 and hence inducedfit might also be applicable to the ligand binding dynamics ofDAs, constituting a complementary binding pathway to con-formational selection. Importantly, induced fit may account for(i) a higher affinity binding pathway with which a DA binds acognate ligand, while also enabling DAs to be modeled as(ii) capable of sensing a cognate ligand even when hybridizedinto a thermodynamically stable duplex by the ACE.

The possibility that DAs might directly recognize and bind aligand from the duplexed state was first considered in a modelby Simmel and colleagues.124,125 Based on kinetic measure-ments of a thrombin-specific DA, however, it was not possibleto assess if the ACE-duplexed aptamer could sense the ligand,nor was a general distinction made between binding pathwaysin describing the behavior of the DA. One of the first reports ona DA also found a small subset of ATP DNA DAs to respond veryfast to ligand (B1 min),32 yet no mechanistic reasoning waspresented as to why these DAs, with ACEs ranging in lengthfrom 12- to 15-mers, responded so quickly, in particular whenconsidering that conformational selection would predict response

Fig. 10 Affinity isoline guided design of solution FRET trans-DAs, alongside aplot of ACE lengths used to engineer DAs in the literature. (a) Under aconformational selection model, the observed affinity of a trans-DA for a ligand(KC.S.

d ) is a function of the predicted ACE–aptamer hybridization affinity (KHyb),the concentration of the ACE ([ACE]), and the known affinity of the nativeaptamer for the ligand (KApt

d ).100 The isolines shown for differing KAptd values can

be used to guide the design of DAs based on aptamers of differing affinities, withACE affinity and concentration chosen for a desired application. Experimentallymeasured affinities of FRET trans-DAs for cocaine99 (blue squares) and ATP100

(magenta circles) are shown, as well as mathematically predicted affinities of theDA systems based on conformational selection (blue and magenta isolines,respectively). (b) Scatter bar plot of the length of ACEs used to engineer DAscompiled from the literature as summarized in Table S1 (ESI†), illustrating thedifferences in ACE length between trans- and cis-DAs. Only perfect matchACEs that were designed to contain no secondary structure were plotted. trans-DAs are shown as green circles, cis-DAs are shown as red squares.


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times on the order of hours or longer, as required for long ACEs todehybridize. To this end, our group recently developed an assayformat to specifically probe induced fit in DAs, revealing theexistence of induced fit in a small family of ATP DNA DAs shownin Fig. 10a.100 Additionally, the location of the ACE in the ATP DNAaptamer sequence was found to regulate the induced fit ligandbinding affinity of these DAs in a non-obvious manner, dependinginversely on the degree of hybridization of the ACE to the ligand-binding pocket of the aptamer – this finding suggests that ACElocation is a key factor affecting DA induced fit ligand binding.

Our group recently developed ACE-Scan121 on microarrays asa method to comprehensively study DA ligand binding internary ACE–aptamer–ligand DA systems, akin to microarrayand sequencing technologies for the study of binary nucleicacid–nucleic acid126–130 and nucleic acid–ligand131–135 systems.ACE-Scan uncovered rich induced fit binding landscapes for DAfamilies engineered from DNA and RNA aptamers against avariety of ligands (ACE-Scan of ATP DNA aptamer shown inFig. 11), highlighting the prevalence of induced fit in DAs.Generally, induced fit binding was promoted by short, 7- to12-mer ACEs hybridizing to bases in the aptamer known tointerface with the ligand. Mismatched ACEs also promotedinduced fit, and this finding may explain the superior performanceof DAs implemented in the literature with singly- or multiply-mismatched ACEs (e.g. ref. 64).

The induced fit model of DA ligand binding was developedbased on experiments conducted by our group, and its wideracceptance will depend on independent corroboration by othergroups along with direct observation of the binding mechanismusing NMR and single molecule FRET experiments that candirectly track structural changes. Whereas the ATP DNA aptameris a good candidate for pilot DA studies, additional studies of newand diverse DA systems is also merited. Additional experimentscould further identify possible limitations of either model,

and clarify critical assumptions of induced fit (e.g. the assumptionsunderlying Michaelis–Menten and/or Briggs–Haldane models ofkinetics), and potentially narrow the scope of induced fit in DAs towithin the range of those assumptions. Future research could alsoleverage the tools and methodologies established for studyingriboswitches to explore the binding dynamics of DAs. Riboswitchesand DAs share many common elements, and as noted in Section 4.1,different riboswitches have been documented over the past decadeto exhibit varying degrees of induced fit and conformationalselection.

Although an induced fit model of DAs is a relatively newviewpoint of DA binding dynamics, it provides a coherent – andintuitive – framework for explaining the high affinity and rapidkinetics experimentally observed for many DA systems. Goingforward, one should also consider that the molecular complex-ity of the induced fit pathway represents a significant challengefor predictive modelers. Induced fit landscapes are highlydependent on the aptamer used to engineer the DA family,and cannot be predicted beforehand based on thermodynamicmodels of ACE–aptamer nucleic acid hybridization.121 As such,universal design rules for the design of ACEs that promoteinduced fit in DAs, which are needed to design DAs withmaximized affinities or for straightforward DA tunability, remainto be elucidated.

4.5 Strand displacement model of DAs

Conformational selection and induced fit models of DA ligandbinding only provide descriptions of the global dynamics ofrepresentative DA states. To model the molecular dynamics ofDA ligand binding, one can apply the principle of nucleic acidstrand displacement to DAs. Strand displacement is a dynamicmodel of the hybridization and dehybridization of nucleicacids, and can be used to describe the kinetics of competitivenucleic acid hybridization, as well as the migration rate of two

Fig. 11 Ligand binding landscapes of ATP DNA DAs generated by ACE-Scan. (a) Hybridizing the ATP DNA aptamer to every 7- to 32-mer ACE generates acomplete hybridization landscape. Here, each tile represents a DA with an ACE of length L that hybridizes starting at 50 position N in the aptamersequence (see inset). (b) Incubation of the microarray with buffer can be used to map the spontaneous dissociation landscape (koff) of surface-assembledDAs. The dataset is shown in 50 (top left) and 30 (bottom right) enantiomeric formats to help visualize the DA dissociation landscape for ACEs thathybridize starting from the 50 or 30 end of the aptamer sequence, respectively. (c) Upon introduction of 10 mM ATP, DA dissociation rates increasesubstantially only for a subset of structurally similar 9- to 12-mer ACEs, identifying these ACEs as hits that promote induced fit (i.e. increased koff,10mM*) inthe ATP DNA DA family. Reproduced from ref. 121 with permission from Springer Nature, copyright 2018.


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nucleic acids that are competing to hybridize to a commonsequence.136–139

Strand displacement was recently applied to model a trans-DA for ATP (Fig. 12).140 ACEs were modeled on the basis oftoehold kinetics, and the ATP-induced intramolecular foldingof the aptamer was modeled as a competitive hybridizationreaction, with the folding aptamer competing directly with theACE for hybridization.140 Under this model, it was assumedthat the ligand binds to the partially unhybridized DA, in linewith an induced fit model of DA ligand binding. The bindingkinetics of the tested DA designs agreed with a strand displace-ment model, with longer ACEs leading to slower binding kinetics.

Strand displacement also provides an intuitive descriptionof the binding dynamics of DAs with very long ACEs (e.g.418mers). By considering that only the portion of the ACEthat is directly hybridized to a primary binding site in the aptamermust first dehybridize before ligand binding can initiate, theexistence of a cooperative effect amongst ligand binding, additionalaptamer folding, and continued ACE displacement would supportmuch faster binding kinetics than a conformational selection-onlymodel in which complete dehybridization is required before ligandbinding.

Although strand displacement could accurately model theATP DNA DA constructs tested by Monserud et al.,140 we notethat a strand displacement model cannot account for the complexbinding behavior previously observed for other constructs in thesame ATP DNA DA family, including the existence of anti-cooperative binding of the two ATP molecules for both cis-141 andtrans-ATP DNA DAs100 engineered with short ACEs, the observationthat small changes in ACE location can non-obviously modulatebinding cooperativities,100,141 as well as the observation thatdecreasing ACE length can lead to the generation of ATP DNADAs that exhibit lower binding affinities.100 This last inconsistencyis of particular interest, as shorter ACEs form DAs with weaker

strand displacement toeholds that are predicted to promote, ratherthan inhibit, DA ligand binding under a strand displacementmodel. As such, at the moment it appears that strand displace-ment kinetics cannot yet account for the non-obvious impact ofACE location on DA ligand binding.100,121

Given the focus of historical DA modeling efforts on con-formational selection, going forward, improved DA ligand bindingmodels would benefit from also taking into account aspects ofinduced fit in tandem with the integration of strand-displacementmodels of ACE dehybridization kinetics. Improved models mayalso have to consider the differences in DA secondary structuresgenerated when using ACEs that vary in location, the kineticsof initial DA–ligand recognition and ligand docking, or three-dimensional time-resolved DA–ligand folding dynamics.

5. In vitro and in vivo applications ofduplexed aptamers

DAs have been used in a variety of in vitro applications for thequantification of diverse ligands. More recently, DAs have alsoemerged as in vivo biosensors in bacterial and mammal modelsystems. Additionally, DA systems have been developed insynthetic biology for sensitive gene regulation applications. Inthis section, we first review the DNA and RNA aptamers mostoften used in the literature to generate DA biosensors. We thencomprehensively review biosensing applications of DAs, covering arange of readout formats. We have divided these applications intosubsections based on the medium in which the DA biosensor wasimplemented, with solution, surface, nanoparticle, and cell-based/in vivo subsections, each of which contain a representative handfulof DA examples with differing readout methods. We have alsocomprehensively summarized the available literature on in vitroand in vivo DA biosensors in Table 1 according to the applicationmode and readout method, the cis- or trans-nature of the DA,whether a DNA or RNA aptamer was implemented, and accordingto the ligand targeted by the DA. Finally, we describe other uses ofDAs and ACEs, as well as natural and artificial riboswitches andsynthetic riboregulators under the framework of DAs and ACEs.

5.1 Model DAs

DAs have been reported in the literature for a number of smallmolecule and protein targets, however a number of DA biosensorconcepts have been engineered using the same handful of aptamers:the ATP DNA, cocaine DNA, theophylline RNA, thrombin DNA, andPDGF DNA aptamers. In this subsection, we briefly describe theaptamers that have been used to engineer these model DAs, asthese aptamers are often chosen to generate DA constructs forproof-of-concept experiments or new assay formats.

DAs are most commonly engineered from the Huizenga andSzostak DNA aptamer against the small molecule ATP142 (Fig. 13a).NMR studies revealed that the ATP DNA aptamer adopts a complexhelical structure that allows it to bind ATP (as well as ADP, AMP andadenosine) with a 6 mM affinity by forming a shared binding pocketthat accepts two ATP molecules,143,144 with the aptamer binding theligands in a highly cooperative manner (Hill slope, n = 2.0).141

Fig. 12 Strand displacement model of ATP DNA DAs. The bindingdynamics of two DA constructs containing 6 (6bT, left construct in A–C)or 11 (11bT, right construct) non-ACE-duplexed aptamer bases are shown.For ACEs with moderate complementarity to the aptamer, as in the 11bTconstruct, the dehybridization of the ACE due to the ligand can bemodeled as a strand displacement reaction, with the ligand-inducedintrastrand folding of the aptamer competing with the interstrand hybridizationof the ACE. For long ACEs, as in the 6bT construct, the increased stability of theACE precludes strand displacement. Bases making up the dual ligand-bindingpocket of the ATP DNA aptamer are colored in red, and ATP is drawn as yellowstars. Reproduced from ref. 140 with permission from John Wiley and Sons,copyright 2016.


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The ATP DNA aptamer has been used in a wide range of DAdesigns, as illustrated in the application examples of this section.

The Landry et al. cocaine DNA aptamer,50 another smallmolecule-binding aptamer, has also been extensively used forDA biosensor designs (Fig. 13b). The cocaine DNA aptamerbinds cocaine, as well as some closely-related molecules,145

with a 500 nM affinity by forming a three-stem planar structurethat binds cocaine at the junction of the stems.

In comparison with DNA aptamers, RNA aptamers have beenused less often to construct DAs, likely owing to the decreasedstability of RNA aptamers arising due to hydrolysis. The mostcommon RNA aptamer used to engineer DAs is the theophyllineRNA aptamer,5 which binds the bronchodilator theophyllinewith 100 nM affinity (Fig. 13c). This aptamer folds into a complexsecondary structure that enables it to exhibit a high degree ofmolecular specificity,146 with the aptamer binding caffeine,which differs by only a single additional methyl group fromtheophylline, with a 10 000-fold lower affinity.5,146

DAs have also been developed for a range of protein targets.The most commonly employed aptamer in DAs targeting aprotein ligand is the thrombin-binding DNA aptamer discoveredby Bock et al. (Fig. 13d).147 This aptamer binds to exosite I on

human alpha thrombin with a 100 nM affinity by forming astacked G-quadruplex structure with two exposed loops.148 Theunique structure, high affinity, and therapeutic potential of thisaptamer has led to the development of a wide variety of thrombinaptasensors, as reviewed elsewhere.149 Interestingly, we note thatanother DNA aptamer, which forms a G-quadruplex and additionalsecondary structure, has been selected to bind to thrombin exositeII,150 giving researchers the potential to use two aptamers againstthrombin in DA biosensor development.

A DNA aptamer against platelet-derived growth factor151

(PDGF-BB homodimer) has also been used as protein-bindingaptamer in a number of DA applications (Fig. 13e). The PDGFDNA aptamer adopts a three-stem structure that allows it tobind PDGF-BB with a 100 pM affinity while maintaining thecapability to differentiate between binding to PDGF homodimervariants (BB vs. AA) by B100-fold.151

5.2 Solution DAs

DAs are most easily implemented in solution applications. Here,the aptamer and ACE sequences are free of spatial constraints,and additional conjugation steps of the aptamer or ACE to asurface are not needed.

Table 1 Summary of in vitro and in vivo applications of DAs for various ligands

Applicationmode Readout method Ligands & references

Solution FRET ATP/AMP/adenosine (ref. 32, 36,a 62,b 68, 103, 163–170a), cocaine (ref. 99, 171), GTP (ref. 68),lysozyme (ref. 170), mercury (ref. 165), ricin (ref. 110b), theophylline (ref. 54,b 172b), thiaminepyrophosphate (TPP) (ref. 54a), a-thrombin (ref. 32, 36,a 53,a 62, 122, 124, 125, 173–176),zinc (ref. 73)

Solution Fluorescence ATP/AMP/adenosine (ref. 38, 177–183a,b), cocaine (ref. 38), GTP (ref. 183a,b), IFN-g (ref. 184),lysozyme (ref. 178), ochratoxin A (ref. 185), PDGF-BB (ref. 186a), potassium (ref. 180),protein tyrosine kinase-7 (PTK7) (ref. 187a), a-thrombin (ref. 180, 181, 188), nucleolin (ref. 189)

Solution Signaling cascade ATP/AMP/adenosine (ref. 154, 182, 190–200a), cocaine (ref. 43, 201), lysozyme (ref. 202a),PDGF-BB(ref. 203, 204, 205a), theophylline (ref. 206a,b), a-thrombin (ref. 195, 207,a 208a)

Solution DNAzyme/ribozyme ATP/AMP/adenosine (ref. 152, 153, 168, 170,a 209a), cocaine (ref. 153), flavin mononucleotide(FMN) (ref. 210b), lysozyme (ref. 152, 170a), Rev (HIV peptide) (ref. 211a,b), a-thrombin (ref. 211)

Sol–gel FRET ATP/AMP/adenosine (ref. 156, 157)Surface FRET ATP/AMP/adenosine (ref. 37, 140, 212), IFN-g (ref. 213), PDGF-BB (ref. 37), phorate (ref. 140)Surface Fluorescence ATP/AMP/adenosine (ref. 100, 121,b 214, 215), adenine (ref. 121b), cocaine (ref. 121),

ethanolamine (ref. 39), IFN-g (ref. 213), mercury (ref. 216), PDGF-BB (ref. 217),a-thrombin (ref. 121)

Surface Electrochemistry ATP/AMP/adenosine (ref. 35, 63, 64, 153, 155, 212, 218–229a), cocaine (ref. 64, 153), IFN-g(ref. 230–232a), IgE (ref. 233a), lysozyme (ref. 224), ochratoxin A (ref. 234), progesterone (ref. 235),theophylline (ref. 236b), a-thrombin (ref. 34, 35, 63, 64, 226, 237,a 238a), TNF-a (ref. 230a,b)

Surface Spectroscopy(plasmon resonance,interference, Raman)

ATP/AMP/adenosine (ref. 40, 239–242), IFN-g (ref. 213), kanamycin A (ref. 71),a-thrombin (ref. 237a)

Surface Signaling cascade ATP/AMP/adenosine (ref. 104, 243), ochratoxin A (ref. 244–246), a-thrombin (ref. 246–250)Surface DNAzyme/ribozyme ATP/AMP/adenosine (ref. 251a)Surface Silver amplification Mercury (ref. 252a), a-thrombin (ref. 252a)Magnetic bead FRET/cytometry/enzymatic ATP/AMP/adenosine (ref. 253, 254), cocaine (ref. 254), IFN-g (ref. 254), kanamycin A (ref. 70, 71),

melamine (ref. 72)Nanoparticle FRET ATP/AMP/adenosine (ref. 41,a 141,a 255, 256a,b), a-thrombin (ref. 257)Nanoparticle Fluorescence ATP/AMP/adenosine (ref. 258, 259, 260a), nucleolin (ref. 261a)Nanoparticle Chemiluminescence Cocaine (ref. 262)Nanoparticle Colorimetry ATP/AMP/adenosine (ref. 42, 158, 169, 263–266), cocaine (ref. 42, 158, 263, 264), potassium

(ref. 263), theophylline (ref. 267b)Nanoparticle Magnetic resonance

imagingATP/AMP/adenosine (ref. 268)

Cells FRET/fluorescence/therapy ATP/AMP/adenosine (ref. 41,a 159, 269–271,a 272c), immunoglobin heavy mu chain (IGHM)(ref. 273a), nucleolin (ref. 189,a 274c), protein tyrosine kinase-7 (PTK7)(ref. 160,a,c 161,a 162,a 275–278,a 279), a-thrombin (ref. 271a)

a Indicates cis-DA design. b Indicates DA engineered with an RNA aptamer. c Indicates in vivo application in a higher organism.


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For solution trans-DAs, FRET readout is often pursued. Asdescribed in Section 2, the first solution FRET DAs were developedby Nutiu and Li, and included a number of candidate DA designsengineered from the ATP DNA aptamer and the thrombin-bindingDNA aptamer (Fig. 14).32 For these applications, first, theconsensus sequence of the aptamer was extended with a short(B5 base) 50 toehold region that was conjugated with a 50-fluoro-phore. To form a DA, this aptamer construct was next hybridizedwith a short, 9 to 15 base-long, 30 quencher-labeled DNA oligo-nucleotide which served as the ACE. Upon addition of ligand to anequilibrium solution of DAs, the ligand-dependent dehybridizationof the DA resulted in a large fluorescence signal gain.

Solution DAs can also be engineered to yield a colorimetricreadout, for example by extending the aptamer sequence with aperoxidase-mimicking DNAzyme and implementing an ACEthat is complementary to both the aptamer and the DNAzyme.In this strategy, ligand-dependent dehybridization of the ACEfrom the aptamer also leads to dehybridization of the ACEfrom the DNAzyme, which allows the DNAzyme to fold into anactive structure that can turnover a colorimetric substrate. Thisstrategy was first used by Willner and colleagues to generatesolution colorimetric DAs targeting AMP and lysozyme (Fig. 15a).152

Subsequently, this group developed an OR logic gate, based onthe dual detection of AMP and/or cocaine, using a tandem-arranged DA with a shared interstrand ACE that contained theDNAzyme (Fig. 15b).153

Whereas FRET and fluorescence DAs often exhibit highsignal gain, DAs are also amenable to even more sensitive methodsthat take advantage of the released ACE. For example, solution DAscan be integrated with nucleic acid signal amplification schemesor a secondary, more sensitive readout step. Qiu and coworkerscombined a DA with a nucleic acid signaling cascade, whichincluded enzyme nicking and a hybridization chain reaction, togenerate a large number of copies of a nucleic acid product inthe first step of a solution assay for adenosine.154 By furtherintegrating a surface electrochemical readout of the amplifiednucleic acid product, this DA circuit achieved fM sensitivity foradenosine in buffer and diluted serum (Fig. 15c and d).154

5.3 Surface DAs

Although the first DA biosensors were developed for solutionassays, DAs are now commonly implemented as surface biosensors,most often using electrochemical readouts. The first surface DAdesigns, termed ‘‘target-induced strand displacement’’ (TID)34

or ‘‘target-induced strand release’’ (TISR),35 relied either ona ligand-binding dependent change in the electron transferproperties of the surface assembled DA (e.g. ref. 34), or on theligand-dependent release of the ACE (e.g. ref. 155) (or alternatively ofthe aptamer–ligand complex (e.g. ref. 35)) from the surface-assembled DA (Fig. 16).

To optimize the signal gain and sensitivity of electrochemicalDAs, charge-neutralizing ACEs (termed neutralizers) were developed

Fig. 13 Secondary structures of aptamers most commonly employed as DA biosensors. Overview of the (a) ATP DNA aptamer, (b) cocaine DNAaptamer, (c) theophylline RNA aptamer, (d) thrombin-binding DNA aptamer, and (e) PDGF-BB DNA aptamer. The ligands and ligand-interfacingnucleotides of the aptamers are colored in green, the aptamer primary sequences are numbered from 5 0 to 30, and important elements of the aptamersecondary structures and ligands are labeled.

Fig. 14 Examples of solution FRET DAs for ATP and thrombin. (a) Overview of assay design. (b) Overview of different DA constructs used for ATP DNAand thrombin DNA DAs. (c) Example of high signal gain obtained for an ATP DNA DA. Adapted from ref. 32 with permission from the American ChemicalSociety, copyright 2003.


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by Kelley and colleagues and used to engineer DAs for thedetection of ATP, cocaine and thrombin (Fig. 17).64 To increasethe sensitivity of these assays, DAs were assembled on nano-structured microelectrodes. Furthermore, ACEs were designedas short PNA strands that contained two single base mis-matches with the aptamer, resulting in lower ACE hybridizationaffinities than a perfect match PNA neutralizer ACE.

In an effort to move away from electrochemical readouts forsurface DAs, Brennan and colleagues demonstrated the first

FRET DA on a glass surface, assembling trans-DAs for ATP andPDGF-BB in which the aptamers were covalently conjugated to aglass surface, and the ACEs were free to diffuse into solutionafter dehybridization (Fig. 18).37 However, the response of theDAs assembled on glass was qualitative only, and not suitablefor quantitative analysis (Fig. 18b). As an extension to surfaceFRET DAs, Brennan and colleagues developed additional DAson sol–gels for small-molecule detection either by incubatingthe preformed sol–gel with the DA (Fig. 18a),37 or by entrapping

Fig. 15 Examples of solution DAs using DNAzymes or signal amplification schemes for readout. (a) DAs specific for AMP or lysozyme that employ DNAzymes.Upon ligand binding, the ACE is released from the aptamer and the DNAzyme. (b) An OR logic gate created using DAs for AMP and cocaine together with aDNAzyme. (c and d) Example of a DA interfaced with a signal amplification scheme. (c) Overview of a solution DA for adenosine, in which ligand binding frees theACE (s1 strand) and initiates an amplified signaling cascade. (d) To increase sensitivity, an electrochemical surface sensor and further signal amplification stepswere used for assay readout. (a) Reprinted from ref. 152 with permission from the American Chemical Society, copyright 2007. (b) Reprinted from ref. 153 withpermission from John Wiley and Sons, copyright 2008. (c and d) Reprinted from ref. 154 with permission from the American Chemical Society, copyright 2015.

Fig. 16 Examples of surface electrochemical DAs. (a) A thrombin-specific DA can be engineered such that signal readout is dependent on the increasedmobility of the ACE after ligand binding, which improves the electron transfer rate. Here, the long toehold on the trans-DA leads it to behave similarly to acis-DA, with a portion of the ACE remaining hybridized to the toehold after ligand binding. (b and c) Surface electrochemical trans-DAs can also beengineered with either the (b) aptamer or the (c) ACE immobilized on the electrode surface, such that ligand binding releases the other element of thetrans-DA into solution, which generates a subsequent change in the electrochemical signal. (a) Reproduced from ref. 34 with permission fromthe American Chemical Society, copyright 2005. (b) Reproduced from ref. 155 with permission from the American Chemical Society, copyright 2006.(c) Reproduced from ref. 35 with permission from The Royal Society of Chemistry, copyright 2008.


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pre-formed DAs within the porous matrix.156,157 In both instances,the sol–gel allows for the ligand to penetrate from the solutionphase, thereby creating a more solution-like environment. As com-pared to DAs engineered on glass, a small improvement in biosensorperformance was obtained when DAs were immobilized on sol–gelsubstrates (Fig. 18c), presumably because these surfaces provided fora more solution-like environment for biosensing, however FRET DAsengineered on surfaces consistently displayed performance inferiorto the same DA design implemented in-solution.37

Scheper and colleagues demonstrated a fluorescence trans-DAon a glass surface coated with a 3D polymer for the detectionof ethanolamine (Fig. 19a).39 In a competitive assay design,the ACE was hybridized to the aptamer simultaneously withligand addition, and the competitive ACE-hybridization and

ligand-binding reactions were allowed to proceed to equilibriumfor 16 h, after which the surface DA was washed and imaged.

In contrast with an equilibrium assay, our group recentlydemonstrated a non-equilibrium DA biosensor on a 2D glasssubstrate (Fig. 19b–e).100 Fluorescence trans-DAs for ATP werefirst assembled on an aldehyde-functionalized glass slide in amicroarray format, allowing for DAs designed with differentACEs to be tested simultaneously (Fig. 19c and d). Following anon-equilibrium protocol, these pre-assembled surface DAswere then queried with ligand for 1 h, after which dehybridizedACEs were washed away and the surface was dried and imaged(Fig. 19c). The fluorescence decreases arising from the incubationof varying ATP concentrations (as compared to buffer only) wasused to generate quantitative binding curves (Fig. 19e), which canbe used to identify the most ligand responsive DA design and todirectly extract the induced fit ligand binding affinity of each DA.

5.4 DAs on nanoparticles

The combination of DAs and nanoparticles affords features ofboth surface and solution assays. Lu and colleagues developedthe first colorimetric DA system based on the disassembly ofgold nanoparticle (AuNP) aggregates in solution (Fig. 20).42,158

First, two oligonucleotides were conjugated to AuNPs. One ofthe oligos acted as an ACE, as it was designed to hybridize to aportion of the aptamer consensus sequence, while the secondoligo was designed to hybridize to an extension of the aptamersequence and act as a tether. Upon addition of aptamer, thehybridization of the ACE and tethering oligos to the aptamerpromoted the aggregation of AuNPs, yielding a blue color. Next,upon addition of ligand, the ACE dehybridized from theaptamer, leading to the disassembly of AuNP aggregates anda change in color to red. This principle was used to construct a‘‘dipstick’’ test, in the form of a microfluidic lateral flow assay,for the detection of adenosine and cocaine.158

Rather than rely on AuNP aggregation, Strouse and colleaguescombined AuNPs with cis-DAs that relied on FRET for readout(Fig. 20).141 The DA was labeled with a terminal fluorescent groupand the other end of the DA was conjugated to the surface ofAuNPs via hybridization to an anchoring oligonucleotide. In thisformat, the AuNP acts as a quencher, resulting in low fluorescencewhen the ACE is hybridized to the aptamer. Upon addition ofligand, DA ligand binding leads to the dehybridization of the ACEand the movement of the fluorophore further away from the AuNP,leading to an increase in fluorescence.

Fig. 17 Example of an electrochemical DA employing a neutralizer ACE.(a) Overview of the electrochemical assay. Here, the ACE is appended withpositively charged amino acids, which neutralize the negative chargespresent on the sugar phosphate backbone of the DNA aptamer. (b) Thehybridization of the neutralizer to the aptamer reduces eT, whereas theligand-induced release of the neutralizer from the DA dramaticallyincreases eT. Reproduced from ref. 64 with permission from NaturePublishing Group, copyright 2012.

Fig. 18 Example of a surface FRET DA on a glass or sol–gel substrate. (a) Overview of FRET DA assay format. (b) Fluorescence response of ATP DNA DAsimplemented on a glass surface. (c) Fluorescence response of ATP DNA DAs implemented on a sol–gel surface. Reproduced from ref. 37 with permissionfrom the American Chemical Society, copyright 2011.


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5.5 In vivo applications of DAs

Although the majority of DAs developed to date have beenimplemented in vitro, more recently, DAs have been developedfor in vivo and intracellular applications in the fields of bio-sensing and biomedicine. One of the first DAs to be applied in acellular organism was termed the nano-flare, consisting of aFRET trans-DA design that was assembled on AuNPs (Fig. 21).41

Because AuNPs are readily taken up by cells, these DA-decoratedAuNPs could be added to live cultured cells, and the resultingfluorescence of cells could be measured to report on intra-cellular metabolite levels. A similar strategy has also beendemonstrated for ATP detection using a cis-DA engineered onlipid micelles.159

In addition to in vivo biosensing applications, DAs have alsobeen applied in biomedical applications. For example, Shi et al.developed cis-DAs against protein tyrosine kinase 7 (PTK7) andapplied them to image tumors in a murine model of cancer(Fig. 22).160 This concept has also been applied to delivertargeted phototherapy to live cells expressing specific surfacemarkers, resulting in increased therapeutic effect.161 DAs thattrigger nucleic acid cascades upon binding to a cell surfaceantigen can also be used to achieve targeted theranostics.162

5.6 Compendium of ligands targeted by DAs

We have summarized the available literature on in vitro andin vivo DA biosensors in Table 1. Besides the examples ofsolution, surface, nanoparticle and in vivo DAs described above,DAs can also be implemented in other assay formats. cis- andtrans-DAs engineered from DNA and RNA aptamers have beenseparated based on application mode, readout method, and

targeted ligand. An additional table with details of each DAdesign is also provided (Table S1, ESI†). While the model DAsdescribed in Section 5.1 have seen widespread implementation,the ATP DNA aptamer and the thrombin-binding DNA aptamerhave been used the most extensively. For example, ATP DAshave been demonstrated for all application modes and almostall readout methods. In general, DAs have been more widelyimplemented in trans than in cis, with DA engineered fromDNA aptamers making up a significant portion of publishedexamples for all DA applications except in vivo studies.

5.7 Other biosensing applications of DAs

Although we have focused our discussion of DAs to cover thoseengineered from ACEs that consist of nucleic acids pre-programmedto hybridize to specific regions of an aptamer, we note that ACEs canalso be developed from other biopolymers, and/or designed tonon-specifically hybridize to the aptamer. The most commonalternatives include single-walled carbon nanotubes (SWNTs)and graphene, both of which form p–p stacking interactionswith single stranded nucleic acids and can be used to immobilizefree aptamer sequences. For example, DAs on graphene- andSWNT that rely on ligand-mediated aptamer–polymer release havebeen demonstrated for in vitro280,281 and in vivo282 applicationswith FRET readout.

In contrast with the use of ACEs to generate ligand-responsiveDA biosensors, as covered in this review, we note that ACEs havealso been engineered to hybridize and stop the binding of anaptamer to its cognate ligand. The straightforward designof strongly hybridizing ACEs to medically active aptamers hasresulted in the development of potent ACE ‘‘antidotes’’ in

Fig. 19 Examples of surface fluorescent DAs. (a) Overview of a signal-off equilibrium assay for the detection of ethanolamine using a fluorescent DA.The ACE competes with the ligand ethanolamine for binding to surface-conjugated aptamers, leading to a ligand-dependent hybridization signal. (b–e)Overview of a non-equilibrium assay using a fluorescent surface DA against ATP. After surface DAs are assembled, the ligand is added and thedissociation rate of ACEs is measured. As shown in (c–e), different ACEs can be implemented in parallel to identify DA constructs that respond directly toligand via induced fit. (a) Reproduced from ref. 39 with permission from the American Chemical Society, copyright 2015. (b–e) Reproduced from ref. 100with permission from The Royal Society of Chemistry, copyright 2017.


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medicine – these ACEs, which are composed of either comple-mentary nucleic acids283,284 or non-specific nucleic-acid bindingpolymers,285 can be administered to reverse an aptamer’s therapeuticeffect. The development of ACE antidotes is of particular significanceto medicine when considering pharmaceutical industry’s increasingefforts to develop drug–antidote pairs.286

Additionally, while this review focuses on DAs engineeredfrom aptamers specific for small molecules and proteins, wehave not discussed or analyzed the design of nucleic acidenzymes (NAEs) in the context of ACEs and DA biosensors.NAEs, which include ribozymes and DNAzymes, are a class offunctional nucleic acid that both (i) bind specific cofactors,which can include heavy metals, small molecules or ions, and(ii) perform an enzymatic action only when bound to thecofactor. NAEs function in an allosteric manner, first bindingthe cofactor, followed by NAE enzymatic action, which mostoften consists of cleaving either a defined cis-located region ofthe NAE or a trans-located ‘‘fuel strand’’ into two fragments.Importantly, NAEs have witnessed significant progress as toolsin synthetic biology and biosensing.28 While outside the scopeof this review, we note that NAEs are also amenable to ACE-specific regulation, as first explored by Burke and colleagues,who used ACEs to tune the enzymatic activity of NAEs, akin tothe design of DAs.287,288

5.8 DAs for gene regulation

DA systems have also been used in synthetic biology for generegulation. Bayer and Smolke demonstrated a gene-regulatingDA design using cis-DAs that served as artificial riboregulators,which upon ligand binding acted in trans to regulate theexpression level of a targeted gene (Fig. 23).289 These DAs weretermed antiswitches, and contained ACEs that were complementaryto both the stem of the aptamer and to a target mRNA sequence.Upon ligand binding, the ACE dehybridized from the aptamer,making it available to bind the target mRNA and inhibit genetranslation. An important factor driving antiswitch design is therelative stability of ACE–aptamer and ACE–mRNA duplexes, andantiswitches can be modeled similarly to the conformationalselection-based design of ACE–aptamer–ligand systems, as describedin Section 4, but by also accounting for the further stabilizationof the ACE-free DA state caused by the hybridization of thefree ACE with the target gene. This DA strategy to engineer

Fig. 20 Example of DA biosensors employing nanoparticles. (a and b) Adipstick employing a DA on AuNPs. Overview of the (a) lateral flow assayand (b) DA constructs used to detect adenosine and cocaine. (c) Exampleof a DA implemented on a nanoparticle that relies on AuNP FRET forsignaling. Four DA constructs were tested, in which the aptamer sequence(highlighted in red) was hybridized in cis- with a 6-mer ACE complementaryto varying positions in the aptamer sequence. Ligand addition resulted inaptamer–ACE displacement and a decrease in FRET. (a and b) Reproducedfrom ref. 158 with permission from John Wiley and Sons, copyright 2006.(c) Reproduced from ref. 141 with permission from the American ChemicalSociety, copyright 2014.

Fig. 21 Nano-flares enable the intracellular quantification of metabolite levels. (a) Overview of FRET ATP DNA DAs on nanoparticles. When the DA ishybridized, the AuNP quenches the fluorescence of the Cy5 moiety on the ACE. High intracellular ATP levels result in ACE dehybridization, leading to anincrease in fluorescence. (b) Nano-flares are readily taken up by HeLa cells, allowing for intracellular ATP levels to be measured. The Cy5 channel (yellow,signaling the concentration of ATP) is shown overlaid the fluorescence of other cellular markers (blue and green). Reproduced from ref. 41 withpermission from the American Chemical Society, copyright 2009.


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gene-regulating ribozyme switches has also been implementedusing cis-acting ACE-hybridized aptamers that transmit ligandbinding events to the ribozyme core on the basis of ACE strand-displacement.290

Interestingly, gene-regulating strategies that employ DAsoften share designs in common with natural riboswitches.Riboswitches are cis-acting RNAs that contain (i) an aptamerdomain and (ii) a modular RNA expression domain, and thesedomains interact in vivo via partial hybridization.291–293 Inriboswitches, ligand binding of the aptamer domain regulatesthe dehybridization of the expression domain, which is ultimatelyresponsible for effecting transcriptional or translational control(Fig. 24). Indeed, this structure lends itself well to the concept of aDA system, in which the ACE corresponds to the natural cis-actingexpression domain.

The prevalence and diversity of natural riboswitches inspiredthe development of artificial riboswitches that make use ofin vitro-selected aptamers to regulate mRNAs transcription ortranslation. For example, using cis-located RNA ACEs to block a

Fig. 22 Example of a cis-DA used for in vivo imaging. (a) Overview of the design of the FRET cis-DA. Upon recognition and binding of the aptamer to thetarget protein on a cancer cell, the ACE dehybridizes from the aptamer, resulting in an increase in fluorescence. (b) The DA binds to its target ligandin vivo, and can be used to locate and image tumors in mice 60 min after DA delivery. Reproduced from ref. 160 with permission from the NationalAcademy of Sciences, copyright 2011.

Fig. 23 Antiswitch riboregulator based on a DA design. (a) Overview of antiswitch mechanism of action. Upon introduction of the ligand (green circle,termed ‘‘effector’’), the DA releases the cis-hybridizing ACE, which allows the ACE to hybridize to an mRNA target, inhibiting translation. (b) Schematic ofone of the antiswitches. Predicted stabilities of the aptamer–ACE and ACE–mRNA duplexes were used to guide antiswitch riboregulator design.Reproduced from ref. 289 with permission from Nature Publishing Group, copyright 2005.

Fig. 24 The xpt guanine riboswitch effects gene regulation in response tointracellular changes in guanine concentration. In the absence of ligand, astable duplex is formed between the aptamer and the expression domain.Upon ligand binding by the aptamer domain (boxed), a portion of theexpression domain (in red, acting as an ACE) is dehybridized from itscomplementary region in the aptamer (cyan) and effects transcriptional ortranslational control of a downstream gene in the mRNA. In riboswitches,the aptamer and expression domains are highly modular, and a number ofconfigurations exist that can either facilitate or inhibit RNA transcription orgene translation upon ligand binding. Adapted from ref. 294 with permissionfrom the National Academy of Sciences, copyright 2005.


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portion of the natural aptamer, Ogawa artificially engineered atranslation-regulating riboswitch that responded to theophyl-line in vitro, and based on the same cis-DA design, riboswitchescould be engineered that also responded to FMN, tetracyclineand sulforhodamine B as ligands.295 Similarly, Hanson et al.developed a theophylline-dependent transcription-regulatingsynthetic riboswitch using a rational design based on the tuningof the hybridization affinity between the ACE and the expressiondomain.44

However, the rational design of optimized synthetic ribo-switches is not straightforward, as many binding reactions andpartners must be considered, including those between theaptamer and the ligand, the ACE and the expression domain,as well as the impact of transcriptional or translational machin-ery on in vivo riboswitch function. Recently, Salis and colleaguesdeveloped an automated method to design synthetic translation-regulating riboswitches on the basis of DAs (Fig. 25).296 In thismethod, the degree of ACE hybridization with the aptamer,including ACE length and the incorporation of mismatches, istuned to ensure that ligand binding leads to sufficient stabilizationof the ACE-free state, while also taking into account the interfacingof the ribosome with both the ribosome binding site (RBS) and thestart codon in the mRNA sequence.

6. Summary and outlook

Over the past 25 years, aptamers have emerged as highlyconfigurable ligand-specific receptors compatible with multiplebiosensing strategies. One of the most widespread aptasensingformats is the DA, in which an aptamer is hybridized with anACE, either in cis- or in trans-, to form a ligand-responsive switch.Importantly, DAs leverage the straightforward programmabilityinherent to all nucleic acids, affording them excellent general-izability across aptamers, as well as high degrees of versatility andtunability. Whereas DAs were first demonstrated for the solution-phase biosensing of small molecules and proteins, DA designshave since been leveraged for a wide range of biosensingstrategies in vitro and in vivo, and also used as gene-regulatingelements in synthetic biology.

DAs have a number of advantages over other aptasensorformats, including a relatively simple design applicable to DNA orRNA aptamers of any sequence, high signal gain, the capability of

being implemented as cis- or trans-DAs as desired, and acompatibility with various readout methods. DAs have limitations,however, both with respect to other aptasensor formats and withrespect to other biosensing strategies. Most importantly, owing tothe competitive binding between ACEs and ligands for the apta-mer, DA biosensors exhibit lower affinities than native aptamers.DAs also rely on the existence of aptamers for the target of interest,and are thus subject to the same limitations with regard to liganddiversity as aptamers. Similarly to native aptamers, DAs are liableto nuclease-mediated degradation, which can lead to false-positive or false-negative signaling unless compensated for bydesign. Additionally, DAs must be used with compatible orvalidated buffers, which are often aptamer-specific, therebylimiting DA multiplexing. In our review of the DA literature,we also note that most reports on DAs do not provide measure-ments of the affinity of DAs for their cognate ligand (Table S1,ESI†). Although mention of linear dynamic range and lowerlimits of detection (LODs) are common, full dynamic rangebinding curves are rarely presented. In this regard, it wouldbenefit the DA biosensor community to follow the reportingstandards commonly employed in the immunoassay field.

A critical limitation to engineering DAs de novo remains ourlimited knowledge of the ligand binding dynamics of DA systems.The conformational selection model, which is widely used foranalyzing DA biosensors and guiding their design (includingaffinity and dynamic range) cannot adequately account for theequilibrium and fast binding kinetics of many DAs, especially incases involving long ACEs, with observed affinities up to onemillion-fold higher than predicted. The conformational selectionmodel seemingly provides a lower bound for binding perfor-mance. The induced fit model that was recently introducedprovides a mechanism to account for the high affinities observedin multiple experiments, and agrees with the recently proposedstrand displacement model of DA systems. Additional indepen-dent verification and mechanistic studies (using NMR andsingle molecule FRET) will help establish the scope and validityof induced fit, as well as its coexistence and relative importancecompared to conformational selection for various DAs. Today,the selection and optimization of DAs and ACEs is typically donevia trial and error through experimentation. From a designoptimization standpoint, the behavior of DAs based on ligandbinding via conformational selection can be readily modeledand predicted, however induced fit binding, and the ACEs that

Fig. 25 Automated design of riboswitches guided by the principle of DA conformational selection. (a) In the off state, the aptamer (sequence in blue) isdesigned to be hybridized by the cis-located ACE, which results in the sequestering of RBS and start codon. (b) Upon addition of ligand, the aptamerdomain is stabilized by binding to the ligand and the ACE is dehybridized, leading to the increased availability of the RBS and start codon, directly leadingto an increase in gene expression. Reproduced from ref. 296 with permission from Oxford University Press, copyright 2016.


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optimally promote induced fit, are not currently predictablea priori. This limitation motivates the exploration and betterprediction of induced fit in DAs engineered from aptamersagainst diverse ligands, and for as many ACEs designs aspossible. In this effort, the search for optimized DAs shouldinclude the testing of mismatched (and other modified) ACEdesigns, as these form DA families in which conformationalselection and induced fit ligand binding pathways might beindependently modified, thereby offering a degenerative meansto engineer DAs with optimized binding properties.121 Likewise,any additional properties of DA performance that might betunable based on ACE design, such as ligand specificity, meritadditional study. Furthermore, we note that DA bindingmechanisms mirror well-known SN1 and SN2 nucleophilic sub-stitution reactions, and the parallels between DAs and thisfundamental class of chemical reactions may both guide thestudy of binding mechanisms, and serve as an enticing entrypoint for organic chemists interested in functional nucleicacids. With SN1 and SN2 reactions in mind, future DA experi-ments could be designed to quantify the contribution of eachrespective pathway (conformational selection vs. induced fit),characterize intermediate energy states, and to study the effectof different solvents, ligand modifications, or perturbations tothe substrate and leaving groups (i.e. to the aptamer and ACE,respectively) on DA binding dynamics. The interplay betweenconformational selection and induced fit ligand bindingmechanisms have also been extensively studied and discussedfor natural riboswitches.98 Given the inherent DA-like functionand design of riboswitches, the growing literature on riboswitchesmay serve to guide future studies of DA binding dynamics asefforts are made to rationally optimize the affinity and dynamics ofDA systems.

Improving our understanding, modeling and control of DAsshould also encourage the adoption of DAs in other nucleicacid nanotechnologies. For example, DAs are poised to be morewidely integrated as ligand-responsive elements in autonomous,pre-programmed nucleic acid circuits.24,139,297 Likewise, DAs arewell-positioned to be incorporated into larger-scale nucleic acidsassemblies, including in origami and nanocages,298 in diagnosticapplications that interface with nucleic acid circuits,299 andin programmable smart complexes300 that are compatiblewith a range of biosensing and medical applications.301,302

Interestingly, improving our understanding of DAs may alsotranslate to the development of improved or novel SELEXworkflows, perhaps offering the potential to quickly and cost-effectively evolutionarily select for the best aptamer–ACE pairfor any ligand(s) of interest, simultaneously in the same SELEXexperiment.

We foresee DAs, and in particular cis-DAs, gaining tractionas versatile biosensing platforms, especially within the analyticalchemistry and biomedical fields. For example, cis-DA biosensorsmay prove to be a more generalizable format for continuousmetabolite monitoring than existing electrochemical aptasensorsbased on modified aptamers,303,304 as DAs do not require carefultuning of an aptamer’s native sequence to yield an acceptableelectrochemical response upon ligand binding. DAs are also

growing in popularity as tools for bioimaging and for targetedin vivo therapies, applications that are simplified when usingsingle-stranded cis-DAs. Going forward, biomedicine will continueto see the transition of DAs from in vitro applications to in vivoconditions,305 the integration of DAs with additional imaging andtherapeutic technologies,306 the further development of DAs fordrug delivery,189 and the development of DAs for novel applications,for example in the generation of antibacterial compounds oridentification of drug targets.307

The concept of DAs as controllable regulatory elements alsostands to be more widely implemented in synthetic biology, andthe utility of induced fit could provide untapped opportunities fordesign. For example, DA systems are suitable as recognition andcontrol elements in the emerging field of riboregulation,308–310

either as trans-activating systems that build on antiswitchregulators,289 or as programmable cis-activating systems that buildon ribozyme regulation.290 The establishment of DAs as general andrationally tunable RNA components would also provide syntheticbiologists with a new, and much needed, toolbox for the allostericregulation of biological functions.86,311

Finally, given the complexity of the induced fit landscapesthat underlie ligand binding in some DA families (as revealedby ACE-Scan), together with the many parallels between DAsand nucleophilic substitution reactions, we note that DAs maybe of significant interest to researchers in the fields of mechanisticmodeling and nucleic acid folding. Given their nucleic acid basis,DAs are highly tractable and configurable, relatively inexpensive toprocure, and tolerant to a wide range of experimental conditions,making them excellent constructs for systematic testing. AlthoughDA systems represent a substantial change to protein modelersand organic chemists, the general concept of partially blockingaptamers with ACEs is a method of system perturbation that isunique to DAs, with different ACEs serving to modify the thermo-dynamics and binding dynamics of the system. For these reasons,DAs may serve as excellent modeling testbeds alongside ribo-switches and other ligand-binder systems, such as intrinsicallydisordered proteins (IDPs), whose complex binding dynamicsare receiving increased attention.312–314

In summary, DAs can be configured into biosensor designsthat can be tailored to the intended application of interest, withthe potential to target a wide variety of ligands. DAs also makeexcellent systems with which to develop in vivo biomedicaltechnologies. Although DAs exhibit rich binding landscapesthat somewhat limit their rational design, this complexity alsomakes DAs attractive from a theoretical and modeling stand-point, and in this sense DAs may serve as unique constructs forbasic science studies. Building on advancements in the modelingand the design of DA biosensors to date, DA systems are poised tocontinue to witness swift progress, both as replacement and ascompanion systems, for a wide range of technologies andapplications in the near future.

Conflicts of interest

There are no conflicts to declare.


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Acknowledgements

We thank Profs. Robert Sladek, Hanadi Sleiman (McGill University)and Alexis Vallee-Belisle (Universite de Montreal) for helpfuldiscussions, and members of our research lab for feedback.This work was funded by the Natural Sciences and EngineeringResearch Council of Canada (NSERC), the Canadian Institutesof Health Research (CIHR), the India-Canada Centre for InnovativeMultidisciplinary Partnerships to Accelerate Community Transfor-mation and Sustainability (ICIMPACTS), and the Canada Foundationfor Innovation (CFI). J. D. M. was supported by an NSERC CanadaGraduate Scholarship. D. J. acknowledges support from a CanadaResearch Chair in micro and nanobioengineering.

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