Structured DNA Aptamer Interactions with Gold Nanoparticles

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Structured DNA Aptamer Interactions with Gold Nanoparticles Peter A. Mirau, Joshua E. Smith, Jorge Luis Chávez, Joshua A. Hagen, Nancy Kelley-Loughnane, and Rajesh R. Naik Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02449 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Structured DNA Aptamer Interactions with Gold Nanoparticles Peter A. Miraua, Joshua E. Smithb, Jorge L. Chávezc, Joshua A. Hagenc, Nancy KelleyLoughnanecand Rajesh Naikc a

Air Force Research Laboratories, Materials and Manufacturing Directorate, Wright-Patterson

AFB, OH 45433 b

Department of Chemistry, Alverina University, Reading, PA, 19607

c

Air Force Research Human Effectiveness Directorate, 711th Human Performance Wing,

Laboratory, Wright-Patterson Air Force Base, OH 45433, United States [email protected]

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) TITLE RUNNING HEAD: A uNP Binding Aptamer in Colorimetric Sensors

ABSTRACT

DNA aptamers that bind biomolecular targets are of interest as the recognition element in colorimetric sensors based on gold nanoparticles (AuNP), where sensor functionality is related to changes in AuNP colloidal stability upon target binding. In order to understand the role of target binding on DNA-AuNP colloidal stability, we have used high-resolution NMR to characterize the interactions of the 36 nucleotide cocaine-binding aptamer (MN4) and related aptamers with AuNPs, cocaine and cocaine metabolites. Changes in the aptamer imino proton NMR spectra with low (20 nM) concentrations of AuNP show that the aptamers undergo fastexchange adsorption on the nanoparticle surface. An analysis of the spectral changes and the comparison with modified MN4 aptamers shows that the AuNP binding domain is localized on

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stem two of the three-stemmed aptamer. The identification of an AuNP recognition domain allows for the incorporation of AuNP binding functionality into a wide variety of aptamers. AuNP-induced spectral changes are not observed for the aptamer-AuNP mixtures in the presence of cocaine, demonstrating that aptamer absorption on the AuNP surface is modulated by aptamer-target interactions. The data also show that the DNA-AuNP interactions and sensor functionality are critically dependent on aptamer folding. KEYWORDS: Aptamer, Au nanoparticles, NMR, colorimetric sensors

Biosensors based on gold nanoparticles (AuNPs) are of great interest because the large changes in surface plasmon resonances that accompany target recognition lead to color changes that are visible to the naked eye.1-3 Although AuNPs do not specifically recognize biomolecular targets, high sensitivity detection schemes have been developed by combining the AuNPs with biological recognition elements (BREs).3, 4 The BREs are constructed from short peptides, DNA or RNA sequences that bind the target substance with great sensitivity and specificity and modulate the colloidal stability of AuNPs, resulting in color changes. 2, 3, 5


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Scheme 1. The aptamer-based colorimetric assay. AuNPs are under consideration as the active element in colorimetric sensors (Scheme 1) because the isolated AuNPs are red in solution and turn purple as the NPs aggregate. Bare AuNPs are not stable in solution, and the rates of aggregation and flocculation are very sensitive to the passivating surface layer, ionic strength, pH and temperature.4, 6 The passivating layers of citrate and surfactants from the synthesis, and peptides, proteins or nucleic acids in sensors, are not static, but are exchanging on a time scale that may play a critical role in aggregation kinetics and sensor response. The color change also depends on the NP size, reactant concentration and order of addition (target added to aptamer-DNA solutions vs. the aptamer-target complex added to AuNP solutions). Biomolecules, including DNA2 and proteins,7, 8 have been observed to adsorb on AuNPs and Au surfaces. The early studies showed that single-stranded DNA (ss-DNA) adsorbs orders of magnitude more strongly than double-stranded DNA (ds-DNA) to AuNPs,2 and it has subsequently been shown that the binding affinity and kinetics depends on the nucleotide (A >>


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C > T) and length.2, 9 The differences in ss- vs. ds-DNA binding have been related to differences in flexibility,2 since the more flexible ss-DNA is able to stabilize the AuNPs by adsorbing the nucleobases to the surface while exposing the charged phosphate moieties to the solvent. This flexibility is not available for nucleotides hydrogen bonded in the ds-DNA helix.10 The fundamental basis for using DNA aptamers in AuNP sensors is that target binding alters the DNA surface adsorption and colloidal stability (Scheme 1). The differences in ss- vs. ds-DNA AuNP adsorption was used in one example to design colorimetric sensors for DNA hybridization.2 The Fan group recognized that this concept could be extended to aptamers that undergo conformational changes in the presence of a target, and a successful colorimetric sensor for K+ was designed using G-quartet forming thrombin binding aptamer.5 Based on these observations and subsequent publications, it has been proposed that a conformational switch from an Au-adsorbing ss-DNA-like state to a folded and base paired ds-DNA-like non-adsorbing conformation in the presence of target is necessary for functional AuNP colorimetric sensors.11 Although DNA aptamers have been used as the recognition element in sensors, surprisingly little is known about the molecular level interactions of DNA and metal NPs.12, 13 The most studied aspects of the DNA-AuNP interactions are the adsorption kinetics and thermodynamics of ss-DNA binding to AuNPs. These studies are most commonly performed using centrifugation4 to isolate the DNA-AuNP complex or the fluorescence quenching of dyelabeled DNA by AuNP absorption.14 These experiments measure the association on a time scale of tens of seconds to minutes, and the desorption on a time scale of tens of minutes.4 Since these assays probe binding on the seconds and longer time scale, any more rapid adsorption processes may escape detection. There is some controversy regarding the interactions leading to DNA adsorption, with some groups concluding the process is dominated by hydrophobic interactions,14 while others conclude it is driven by hydrophilic interactions.4 Since the adsorption mechanism is a subject of controversy, extrapolating the behavior of ss-DNA and dsDNA to aptamers is not a trivial matter. The interactions of aptamers with AuNPs are particularly complex because they can exist as partially folded structures with loops and doublestranded stems that fold into more compact three dimensional structures15 upon target binding that are not representative of either ss-DNA or ds-DNA. Furthermore, the sensing mechanism proposed to explain the functionality of colorimetric sensors, that an unbound aptamer adsorbs


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readily on the AuNP surface but a bound aptamer does not, has not been experimentally confirmed and has only been inferred from the colorimetric response. In these studies we report on the interactions of aptamers with AuNPs and targets in order to understand how aptamer folding and adsorption impacts the colorimetric assay. To probe these interactions at the molecular level, we recorded the Nuclear Magnetic Resonance (NMR) spectra of cocaine binding aptamers in the presences of AuNPs, cocaine and cocaine metabolites. The 36-nucleotide (nt) cocaine-binding aptamer (MN4) has been identified as a strong binder to cocaine (Kd=7±1 µM) but not cocaine metabolites,16 and subsequent studies have shown that the binding affinity depends on the nucleotide sequence.17 The structural studies show that MN4 folds into a three-stem structure with the cocaine binding site at the junction of the three stems.17 Although MN4 folds into a 3 stem structure in the absence of cocaine, a more stable folded structure is thermodynamically favored in the presence of cocaine. Our results show that NMR is a powerful method to study aptamer-AuNP interactions that may not be observable by other methods, and we report on specific interactions between one stem of the folded aptamer and the AuNP surface. The NMR studies of aptamer folding in the presence and absence of AuNPs allows us to probe the role of aptamer folding in sensors and to understand how manipulation of external variables (temperature and ionic strength) can improve the colorimetric response. Our hope is that expanding these studies to aptamers with different structural features and other nanomaterials will guide improved sensor design.

RESULTS AND DISCISSION Folded DNA and RNA aptamers with high affinity and selectivity for target molecules can be identified from random nucleotide libraries18, 19 and are of great interest as the active element in sensors. Aptamers typically contain DNA or RNA sequences with loops and stems stabilized by base pair formation that may further fold into compact structures15 to create binding pockets for small molecules, including cocaine,20 arginine,21 theophiline22 and cortisol. 23 The mechanism and kinetics of target capture and target-induced aptamer conformational change are critical for the rational design of aptamer-based sensors. Much of our understanding of sequence-structure relationships and aptamer folding patterns is derived from NMR 15 and X-ray24 structure determination. The guanine N1 and thymine N3 imino protons are protected from solvent exchange by base pair formation and


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folding, and can be observed by NMR in the region between 10-15 ppm.25, 26 The AT and GC base pairs typically appear in the range of 13-14 and 12-13 ppm, and are well resolved from the exchangeable protons in the noncannonical GA and GT base pairs that appear at higher field.27, 28 The three-dimensional structures of aptamers can be determined from the chemical shifts, coupling constants and the nuclear Overhauser effects between the exchangeable imino and amino protons and the non-exchangeable base and sugar protons.15 It is well documented that the NMR chemical shifts, line widths and relaxation times depend on the dynamics of the system under study.29 The dynamic processes that can affect the spectral appearance in our system are the imino proton exchange rates, the kinetics of the aptamer absorption on AuNPs and the kinetics of aptamer target recognition. The NMR exchange limit is defined by the exchange rate (kex ) relative to the difference in chemical shift (∆ω) between the two states.29 In the slow exchange limit (kex > ∆ω), and broadened lines are observed in the intermediate exchange limit (kex ≈ ∆ω). The imino protons are shifted by about 10 ppm from the water signal (4,000 s-1 at 400 MHz) so exchange rates faster than this will appear at the averaged chemical shift. Since the water is present at much higher concentration than the aptamer (55 M vs. 200 µM), the population-weighted fast-exchange peak position is too close to the water peak to be resolved. The intrinsic imino exchange rate is on the order of 105 s-1,26 so they cannot be observed unless they are protected by base pair formation or aptamer folding. The chemical shift differences for the aptamer protons in the free and target-bound states are likely to be less than 1 ppm, so exchange rates faster than 400 s-1 will be in the fast exchange limit.


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Figure 1. The 400 MHz proton NMR spectra of the MN4 aptamer in the (a) absence and (b) presence of 20 nM 15 nm AuNPs at 298 K.

NMR studies have shown that the 36-nt MN4 aptamer folds into a three-stem structure that undergoes further changes in the presence of cocaine.17

The folded aptamer is stable at low

temperature and melts at 308 K, well above the temperature (298 K) used for the colorimetric cocaine assay. Figure 1(a) shows the 400 MHz imino proton spectrum of MN4 at 298 K in water, where a number of GC and AT base pairs can be identified.17 At low temperature (278 K, not shown) the exchangeable proton signals from the GA base pairs (G29 and G30) can also be observed in the region between 10.5 and 11 ppm. One feature to note in the spectrum at 298 K is that signals from stem 1 (G2, G4, G31 and G34), stem 2 (G9, G10, T15 and T18) and stem 3 (G27, T28) are observed. The peaks from the nucleotides near the 3-stem junction (T18 and G31) are also observed, and well resolved from the other signals. Figure 1(b) shows that large changes in the imino spectra are observed for MN4 (200 µM) in the presence of a 20 nM solution of 15 nm AuNPs in the HEPES buffer used in the colorimetric assay.30 The spectrum is simplified in the presence of the AuNPs as some peaks disappear or decrease in intensity, while large changes in the line widths for the remaining peaks are not observed.


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As noted above, the DNA imino protons are observable when they are protected from solvent exchange by base pairing and/or folding. The possible explanations for the loss in signal intensity in the presence of AuNPs include increased line widths from binding to a 15 nm AuNP in the slow exchange limit, catalysis of imino proton exchange from residual small molecules remaining from the AuNP synthesis or partial aptamer unfolding as a consequence of aptamer adsorption on the AuNP surface in the fast exchange limit. We can eliminate slow exchange binding of the aptamer to the AuNPs as a cause of the spectral changes based on the line widths and stoichiometries. If the aptamers were strongly bound to the AuNPs in the slow exchange limit, much larger line widths would be observed as the hydrodynamic radius would increase from ~ 2-3 nm for the free aptamer to 8-10 nm for the MN4-AuNP complex. Furthermore, the sample contains a large excess of aptamer (200 µM) relative to the AuNP (20 nM). A 15 nm AuNP has a surface area of ~700 nm2 and if we assume that each aptamer has a footprint of 2 nm2, then we could adsorb as many as 350 aptamers per AuNP. Experimentally, however, we measure ~60 aptamers bound per AuNP (Supplementary Information), which corresponds to an aptamer:AuNP adsorption site ratio in excess of 150:1. Any slowly exchanging aptamer would have broadened lines, but the bound fraction would be so small that no significant changes would be observed in the spectrum. Control experiments (Figures 2 and 4) show that increased imino proton exchange is not catalyzed by HEPES buffer, citrate or contaminants remaining from the AuNP preparation and purification. Our hypothesis to explain the spectral changes is that the MN4 adsorbs on the AuNPs surface with a specific geometry in the fast exchange limit, and that this adsorption event promotes partial unfolding of the aptamer, leading to the exchange of selected imino protons. A closer inspection of Figure 1(b) shows the peaks experiencing the greatest change in the presence of the AuNPs are those in stem 2 (T15 and T18) or at the 3-stem junction (G31). The exchangeable amino and nonexchangeable base and sugar protons also show chemical shift changes in the presence of AuNPs (Figure S1), supporting the idea of a structural rearrangement upon adsorption to the AuNPs. To our knowledge this is the first report of the structure-specific interaction of an aptamer with AuNPs.


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Figure 2. The proton NMR spectra of d(GCGCAATTGCGC)2 in the (a) absence and (b) presence of 15 nm AuNPs at 298 K.

In order to determine if the AuNP interactions are common to all DNA sequence or specific to the cocaine-binding aptamer, we measured the NMR spectra of the 12-mer d(GCGCAATTGCGC)2 and modified versions of the MN4 aptamer presence and absence of AuNPs. The 12-mer, which has been extensively studied by X-ray31 and NMR, 32, 33 gives rise to six symmetry-related peaks in the imino proton NMR spectra at low temperature. Figure 2(a) shows that only five peaks are observed at 298 K, since the terminal base pair rapidly exchanges with water and cannot be directly observed. Figure 2(b) shows that no significant changes in peak positions or line widths are observed in the presence of 20 nM AuNPs, although a small peak from the terminal base pair is observable (13.2 ppm) in the presence of AuNPs. This control experiment demonstrates that this linear ds-DNA sequence does not show the same interactions with AuNPs as the cocaine-binding aptamer, and that the buffer and chemicals remaining from the AuNPs preparation do not catalyze imino proton exchange. Other controls are presented in the supplementary materials.


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Scheme 2. Aptamers MN6 and ST13CT derived from MN4.

To further evaluate the AuNP binding domain of MN4 we considered several modifications of MN4, including MN6 and ST13CT (Scheme 2). MN6 is a previously reported aptamer17 created by removing three base pairs from stem 1 and replacing C23 with T to form a GT base pair, which lowers both the stability and the cocaine affinity. The ST13CT aptamer is the modified MN4 in which stem 2 is removed by cutting between A7-A8 and T18-T19 and connecting A7 to T19. The bridging CT was retained to provide sufficient flexibility to allow for GA base pair formation and provide a binding pocket for the target. Both modifications retain the GA base pairs at the central junction.


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Figure 3. The imino proton spectra of MN6 in the (a) absence and (b) presence of 20 nM AuNP at 278 K.

Figure 3 compares the imino proton NMR spectra of MN6 in the absence and presence of AuNP at 278 K. The AuNP binding was measured at 278 K because removing the three base pairs from stem 1 decreases the stability such that the imino proton spectra of MN6 cannot be observed under assay conditions at 298 K (Figure S3). As with MN4, changes in the chemical shifts and peak intensities are observed in the presence of 20 nM AuNP. This shows that the length of stem 1 is not critical for the fast-exchange binding to the AuNP. The G imino protons from the GA base pairs can be observed in the spectra at 278 K in the region between 10 and 11 ppm. We observe substantial chemical shift changes in the GA base pair protons in the presence of AuNPs, but there is no loss in intensity due to rapid proton exchange.


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Figure 4. The imino proton NMR spectra of ST13CT in the (a) absence and (b) presence of AuNPs at 298 K.

Figure 4 shows the effect of AuNP on the imino proton spectra of ST13CT, the MN4 analog in which stem 2 has been replaced by a bridging CT. In contrast to MN4 and MN6, only minor changes in line width are observed with the addition of AuNP, with no loss of signal or large chemical shift changes. This comparison shows that stem 2 is critical for AuNP adsorption. A similar result is observed for the ST13T aptamer in which stem 2 is replaced with a single bridging T (Figure S3).


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Figure 5. The proton NMR spectra of (a) MN4, (b) MN4/AuNP, (c) MN4/AuNP/Cocaine and (d) MN4/Cocaine at 298 K.

The imino proton spectra are sensitive to the local structure and have been used to study the effect of target recognition on AuNP adsorption (Figure 5). As previously reported,17 significant spectral changes are observed for MN4 in the presence of cocaine, and the data are consistent with the formation of a 1:1 complex. Figure 5(c) shows that spectral changes are also observed for the MN4/AuNP mixture with the addition of cocaine. These changes include shifts in the T imino peaks (13.5-14.3 ppm), and upfield shift for the G31 peak (from 12.6 to 12.1 ppm) on stem 1 nearest the three-stem junction, and the appearance of GA peaks between 10.5-10.8 ppm at the three-stem junction (G29 and G30). Figure 5(c) shows the spectra for the AuNP/MN4/cocaine


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mixture are nearly identical to those observed for MN4/cocaine complex, demonstrating that the MN4/cocaine interaction is much stronger than AuNP/MN4 interaction. Control experiments (Figure S4) showed no change for the MN4 aptamer in the presence of EME, a cocaine metabolite used as a negative control.

Figure 6. The proton NMR spectra of (a) MN6 (b) MN6/AuNP and (c) MN6/AuNP/cocaine and (d) MN6/cocaine at 278 K.

The effect of cocaine on the MN6/AuNP mixture was also explored by NMR. Figure 6(c) shows that large spectral changes are observed for the MN6/AuNP solution with the addition of cocaine. These changes include the appearance and shifting of peaks in the AT, GC and stem junction regions. The spectra are nearly identical to those observed for the MN6/cocaine


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complex (Figure 6(d)), suggesting that, as with MN4, the presence of the aptamer target greatly reduces the interactions of the DNA with the AuNP surface. This shows that the aptamer-target interactions are much stronger than the aptamer-AuNP interactions, and that target binding modulates the interaction of the aptamer with the AuNP.

Figure 7. The (a) colorimetric sensing of cocaine and EME (a cocaine metabolite) using AuNP with the 36 nt MN4 aptamer, (b) comparison of the AuNP/MN6 response to cocaine and EME at 277 and 298 K and (c) the effect of NaCl on the absorbance spectra of AuNP, AuNP/12mer, AuNP/ST13CT, AuNP/MN4 and AuNP/MN6 solutions at ambient temperature. The NMR experiments reported here were motivated in part by the observation that MN4 showed good performance in the AuNP-based colorimetric cocaine assay at ambient temperature while MN6 did not. Figure 7(a) shows a typical assay result for the MN4/AuNP mixture in the presence of cocaine and EME. After observing the folded and unfolded structure by NMR for MN6 at 278 and 298 K, we repeated the colorimetric assay for cocaine and EME at low


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temperature. The results (Figure 7(b)) show that cocaine does not lead to a change in colloidal stability and color change at 298 K, but shows good cocaine sensitivity at 278 K, a temperature at which the NMR data shows that MN6 is partially folded. The NMR and assay results show that aptamer prefolding plays a critical role in the colorimetric assay. While DNA aptamers for sensing applications are typically identified from combinatorial libraries as binders for a target substance, off-target binding is sometimes observed.34 Such behavior has been observed for cocaine binding aptamer where it has been reported that quinine has 30-fold stronger binding affinity relative to cocaine (0.23 µM vs. 7 µM).35 Among the structural differences between cocaine and quinine are the bicyclic aromatic ring in quinine that may lead to more efficient stacking interactions with the DNA base pairs. Cocaine-related molecules and metabolic products are also reported as binders for the cocaine aptamer. Cacoethylene and norcocaine, for example, have affinities similar to cocaine, while 6,7dihydroxycocaine and 6,7-dehydro norcocaine are weaker (5-10x) binders and EME is a nonbinder.36 We anticipate that the binding affinities of off-target substances could have an impact on the NMR results reported here. Changes in the aptamer NMR spectra in the presence of AuNP reflect the relative affinities of the aptamer for the AuNP and the target. We expect similar results for strong binders (like quinine) since the target complex is even more stable35 and the binding should greatly exceed the aptamer-AuNP affinity. As the affinities become weaker there will reach a point where the target affinity is on the same order of magnitude as the aptamerAuNP affinity and more complex NMR behavior may be observed. EME was chosen as a control for these experiments to demonstrate that aptamer binding is required to observe changes in the NMR spectra. We have recently used these off-target interactions to create sensor arrays for the detection of mixtures of compounds.37 The low sensitivity of NMR spectroscopy makes it necessary to perform NMR experiments at aptamer and AuNP concentrations much higher than those used in the colorimetric assay (Aptamer: 200 µM vs. 300 nM; AuNP: 20 nM vs. 5 nM). One consequence of the higher concentration of DNA is that we can only measure the fast-exchange binding by NMR, not the slow-exchange binding measured in the centrifugation38 and fluorescence14 assays. In order to determine the effect of aptamer on AuNP colloidal stability under assay conditions, we measured the salt concentration required to precipitate AuNPs in the presence of MN4, MN6, ST13CT and


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the 12-mer at 298 K. Figure 7(c) show that all of the tested DNAs protect the AuNPs from saltinduced aggregation, but the degree of protection depends on the DNA sequence and structure. The relative order of protection from salt-induced aggregation is MN6 >> MN4 > 12-mer > ST13CT > citrate. The observation that the single-strand like unfolded MN6 offers the greatest protection is consistent with the research showing that single-stranded DNA has a greater affinity for AuNPs than double-stranded DNA. For the folded DNAs, the MN4 shows the greatest interactions with AuNPs and offers better protection than ST13CT and the double-stranded 12mer that showed no evidence of AuNP interactions by NMR. Aptamers have been used in combination with AuNPs to develop colorimetric sensors for a variety of targets,3, 16, 20 and while it is generally accepted that the role of the aptamer is to modulate the colloidal stability by surface absorption, the mechanism and the role of aptamer structure remains open to question. Most of the proposed mechanisms are based on the difference in AuNP affinity for ss- vs. ds-DNA,2, 4, 14 and, with few exceptions,16 the effects of aptamer folding are not explicitly considered. The NMR studies of the MN4 cocaine binding aptamer show that it is folded into a threestem structure in solution, but undergoes additional restructuring to a more thermodynamically stable state in the presence of cocaine.17 The imino proton NMR spectra for both MN4 and the less stable MN6 show large changes with the addition of low AuNP (20 nM) concentrations, when the DNA is present in a 150-fold excess over the available adsorption sites on the AuNPs. Given this stoichiometry, the changes in the aptamer NMR spectra in the presence AuNPs can only result from rapid aptamer exchange at the AuNP surface. Our interpretation is that the loss of imino proton intensity in the presence of AuNPs is due to the exchange of the hydrogen bonded protons with solvent as a consequence of an adsorption event. The observation that specific imino protons are exchanged upon binding shows that the aptamer is not completely unfolded by adsorption on the AuNP surface, rather there is some sequence-specific interaction between the aptamer and the AuNP. The NMR data suggests that stem 2 interacts preferentially with the surface, a hypothesis that is supported by the binding of MN6, which contains stem 2 but a shortened stem 1, and the lack of adsorption for the ST13CT and ST13T aptamers where stem 2 is removed. Since it is well known that the Au binding for A >>T, C2, 14, 39 it is possible that the specific interaction relates to the three A’s making up the loop of stem two next to an adjacent AT base pair. To our knowledge, this is the first reported case of the recognition of a


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folded DNA structure by a metal nanoparticle. This observation makes it possible to consider adding AuNP binding domains to other aptamers, greatly expanding the use of colorimetric sensors based on AuNPs. Furthermore, this specific interaction is modulated by target binding, providing and on-off switch for AuNP interactions.

Scheme 3. A schematic diagram depicting the equilibrium between the folded aptamer, AuNPs and cocaine (shown by the red triangle). The imino protons for the red nucleotides in the aptamer exchange upon surface adsorption. The NMR and colorimetric results are consistent with a model in which the adsorption equilibrium of the folded aptamer plays a critical role in the sensor response. Scheme 3 shows a drawing of the equilibrium between the free and absorbed aptamer that results in imino proton exchange for the nucleotides in red. The binding to cocaine is much stronger than AuNP adsorption, so the equilibrium is shifted to the target-bound state in the presence of cocaine. This modifies the AuNP surface, resulting in aggregation and the colorimetric response. Nonbinding targets, including EME, do not alter this equilibrium and do not affect the colorimetric response. A key part of our hypothesis is that the colloidal properties of AuNPs are critically dependent on the fast-exchange adsorption of the folded aptamer. This fast exchange adsorption is deduced from changes in the NMR spectra that may not be detectable in centrifugation4 or


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fluorescence9 assays that are sensitive to binding on a longer time scale. The salt-induce precipitation experiments show that the folded and unfolded aptamers as well as the linear dsDNA 12-mer all adsorb on AuNPs, but the degree of protection from salt-induced aggregation depends on the folded structure and nucleotide sequence. The difference in the colorimetric response for linear DNA and the aptamers demonstrates that folding is essential for sensor performance. The poor performance of the MN6 in the colorimetric assay at 298 K is attributed to very strong ss-DNA-like binding for the unfolded aptamer that makes the MN6 aptamer unable to recognize and bind the target in the AuNP-bound state. Furthermore, the rapid color change observed in the sensors suggests that the rapid exchange kinetics could be more important for sensor performance than the very slow, strong binding measured in the centrifugation and fluorescence assays.

CONCLUSIONS

The mechanism of AuNP aggregation is critical for understanding and predicting colorimetric sensor performance using biological recognition elements. In these studies we have used NMR spectroscopy to probe the molecular interactions at the AuNP surface that affect colloidal stability and color change in colorimetric assays. The high resolution in the NMR experiments allows us to identify specific interactions between the aptamer and the AuNP surface. The identification of specific interactions makes it possible in principle to incorporate AuNP binding domains into a wide variety of aptamers to greatly expand the range of colorimetric sensors. The comparison of the NMR and colorimetric response shows that the folded structures are critical for sensor performance, and that the adsorption of DNA sequences on AuNPs is not sufficient to give good performance in target-based assays. High-resolution NMR has made enormous contributions towards understanding structure-function relationships in proteins and DNA, and we are optimistic that these tools can be applied towards optimizing colorimetric sensor design using aptamers and other biological recognition elements. Supplementary Information: Additional materials and details of the methods are included in the supplementary information. Materials and Methods Materials:


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Analytical grade chemicals were purchased and used without purification. DNA sequences (MN4: 5’- GGC GAC AAG GAA AAT CCT TCA ACG AAG TGG GTC GCC - 3’; MN6: 5’ - GAC AAG GAA AAT CCT TCA ATG AAG TGG GTC - 3’; 12-mer: 5’-GCG CAA TTG CGC; ST13CT: 5’- GGC GAC ATC AAC GAA GTG GGT CGC C -3’; ST13T: 5’- GGC GAC ATA ACG AAG TGG GTC GCC -3’) were acquired from Integrated DNA Technologies, Inc. (Coralville, IA) and purified by HPLC. Cocaine and ecgonine methyl ester hydrochloride (EME) standards were received from Lipomed as 1 mg/mL methanol solutions. The 15 nm AuNP were prepared as previously described.30 Nuclear Magnetic Resonance: The AuNP were concentrated to 100 nM using tangential flow filtration and mixed with DNA and cocaine in 10 mM HEPES and 1 mM MgCl2 at pH 7.4. The final DNA and AuNP concentrations in all experiments were 200 µM and 20 nM, respectively. Proton NMR spectra were obtained on a Bruker 400 MHz or 800 MHz Avance NMR spectrometer in 90:10 H2O:D2O mixtures using the W5 water suppression pulse sequence 40 with temperature regulation at ± 1 o

C.

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