Modulating Fluorescence Anisotropy of Terminally Labeled Double

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Modulating fluorescence anisotropy of terminally labeled dsDNA via the interaction between dye and nucleotides for rational design of DNA-recognition based applications Hongduan Huang, Hejia Wei, Mingjian Zou, Xiao Xu, Bin Xia, Feng Liu, and Na Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504028n • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Modulating fluorescence anisotropy of terminally labeled dsDNA via the interaction between dye and nucleotides for rational design of DNA-recognition based applications Hongduan Huang[a], Hejia Wei[b,c], Mingjian Zou[a], Xiao Xu[a], Bin Xia[a,b,c]*, Feng Liu[a] and Na Li[a]* [a] Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China. [b] Beijing NMR Center, Peking University, Beijing 100871, China. [c] College of Life Sciences, Peking University, Beijing 100871, China. ABSTRACT: Effective signal enhancement for fluorescence anisotropy in a simple manner is most desirable for fluorescence anisotropy method development. This work aimed to provide insights into the fluorescence anisotropy of terminally labeled doublestranded DNA (dsDNA) to facilitate a facile and universal design strategy for DNA-recognition based applications. We demonstrated that fluorescence anisotropy of dsDNA could be regulated by the nature of dyes, the molecular volume, and the end structure of dsDNA. Fluorescence anisotropy ascended with the increased number of base pairs up to 18 bp and leveled off thereafter, indicating the molecular volume was not the only factor responsible for fluorescence anisotropy. By choosing dyes with the positively charged center, high fluorescence anisotropy signal was obtained due to the confinement of the segmental motion of dyes through the electrostatic interaction. By properly designing the end structure of dsDNA, fluorescence anisotropy could be further improved by enlarging the effective overall rotational volume, as supported by 2D 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY). With the successful enhancement of the fluorescence anisotropy for terminally labeled dsDNA, simple and universal designs were demonstrated by sensing of major classes of analytes from macromolecules (DNA and protein) to small molecules (cocaine).

Fluorescence anisotropy (FA) has been frequently used in therapeutic drug monitoring and screening, detection of allergens, drugs and toxins in food, as well as binding assays such as protein-protein and enzyme-inhibitor interactions.1-8 However, the breadth of applications of fluorescence anisotropy methods based on DNA-recognition has been impeded by the difficulty in simply and efficiently enhancing the fluorescence anisotropy signal of terminally labeled double-stranded DNA (dsDNA), which has most frequently been involved in binding assay and biosensing purposes, especially for small molecules.9-24 Currently the strategies mainly focus on the molecular volume or mass by using DNA-binding proteins or nanoparticles, which complicates the experimental design and manipulation; and the signal often suffers from scattered light from nanoparticles.11,13,14,17-20,22,25 Yet, the fluorescence anisotropy has been found not to always increase with the length of dsDNA or the molecular weight of the formed complex,15,26 suggesting the limited effect of volume factor on signal enhancement. Besides, the binding induced conformational change strategy strongly requires the customized design of the DNA sequence as well as the secondary/tertiary structure, which cannot be easily realized for universal experimental designs.16 Although the competitive assay based on stranddisplacement has achieved successful detection of small molecule, e.g. ochratoxin A (Kd=50 nM),27 there is no design rationale provided in terms of further altering the fluorescence anisotropy, thus applications to small molecules with affinity

toward the aptameric probe (Kd at micromolar or millimolar level) weaker than that of ochratoxin A may not be feasible. On the other hand, there have been studies showing that the local structural change associated with binding of the fluorophore plays an important role in fluorescence anisotropy,9-12 which could be advantageous for the favorable signal enhancement; however, the mechanism for fluorescence anisotropy signal change still lacks in-depth understanding.28,29 Hence, a comprehensive study of the influence of the nature of dyes and DNA end structure on fluorescence anisotropy of terminally labeled dsDNA is urgently needed to facilitate a facile design strategy suitable for most targets. In the present study, fluorescence anisotropy of short dsDNA labeled with ROX (6-carboxyl-x-rhodamine), which has a positively charged center, was carefully investigated with the assistance of 2D 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) to unravel the underlying mechanism accounting for the fluorescence anisotropy change. Effective improvement of fluorescence anisotropy has been achieved by selecting the appropriate dye, tuning the length of dsDNA and the end structure of dsDNA. The results demonstrated that the dye-DNA interaction, in addition to the molecular volume, is another effective approach for the modulation of fluorescence anisotropy of terminally labeled dsDNA. Based on the successful modulation of fluorescence anisotropy signal, we have proposed a simple and universal design in which the signal

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change associated with target binding is only determined by the short fluorescent DNA probe and its hybridized dsDNA but not involve the target itself, facilitating the facile detection of small molecules. To demonstrate the principle of design, calibration curves have been constructed for major classes of analytes including DNA, protein and especially the small molecule which is difficult to detect using aptamer-based fluorescence anisotropy. This design can avoid using nanomaterials,17-19,22 single-strand11 or double-strand20 binding proteins, enzymes,23 as well as complicated sequence or configuration designs,30 opening a new avenue for fluorescence anisotropy based sensing applications.

Experimental Materials and instrumentation. All the ssDNA were synthesized and HPLC-purified by Sangon Biotech (Shanghai) Co., Ltd. All fluorophores used in this study, 6carboxyfluorescein (FAM) or 6-carboxy-x-rhodamine (ROX) and 5-carboxytetramethylrhodamine (TAMRA), were attached to the 5′-end of the ssDNA. Glycine (AR) and trizma base (AR) were purchased from Beijing Xinjingke Biotechnology Co., Ltd. K2HPO4 (AR), HCl (AR) and other reagents were purchased from Beijing Chemical Works. Wahaha® purified water was used throughout the study. A 50 mM Tris buffer was used throughout the study. The buffer was composed of 50 mM trizma base, 100 mM NaCl, 1 mM NgCl2, and adjusted to pH 7.50 by HCl. Fluorescence lifetime and dynamic fluorescence anisotropy were measured using a Tempro-1 time-correlated singlephoton counting fluorometer (Horiba Jobin Yvon.) with a 590 nm laser source. Instrument response function was scanned with 30% (wt) colloidal silica suspension in H2O. Circular dichroism spectra were obtained with a J-810 spectropolarimeter (JASCO). The 2D 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) spectra were recorded on Bruker Avance 700 MHz spectrometer with a cryo probe at 298 K, with a mixing time of 180 ms and a relaxation delay of 2.0 s. Fluorescence anisotropy measurements. Steady-state fluorescence anisotropy were measured using FLS 920 fluorometer (Edinburgh Instruments Ltd.) with 587.4 nm excitation for ROX, 553 nm for TAMRA and 492 nm for FAM. All fluorescence measurements were carried out with the temperature of the sample compartment maintained by a circulating water bath at 25 ± 0.1 oC unless indicated. Briefly, components of the solution to be tested were added to a 1.5-mL Eppendorf tube to a final volume of 400 µL with concentrations as indicated, and mixed by vortex for 10 s. Fluorescence measurements were carried out after 2 min. The anisotropy, r, of the test solution was calculated by r = (IVVIHH - IVHIHV) / (IVVIHH + 2IVHIHV) where I represents the intensity of the fluorescence signal and the subscript defines the orientation (H for horizontal and V for vertical) of the excitation and emission polarizers, respectively. NMR measurements. The 2D 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) spectra were recorded on Bruker Avance 700 MHz spectrometer with a cryo probe at 298 K, with a mixing time of 180 ms and a relaxation delay of 2.0 s. The spectra were processed using the NMRPipe System31 and analyzed with NMRView 4.1.2.32 Proton chemical

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shifts were referenced to the internal DSS (4,4-dimethyl-4silapentane-1-sulfonic acid). DNA was dissolved in a D2O buffer containing 20 mM Na2HPO4/NaH2PO4, 100mM NaCl and 1 mM MgCl2 (pH 7.0), and the final concentration of DNA was 1 mM. 2D 1H-1H NOESY spectra were obtained and NMR resonance assignments (for protons H1’, H2’, H2’’, H3’, H6/H8, H5/M) were carried out based on the NOESY data. Resonances from nucleotides in dsDNA region were confidently assigned, but chemical shifts of the flexible overhang residue atoms were difficult to determine. T25 and T26 were assigned unambiguously while some resonances were ambiguously assigned as intra-nucleotide NOE from C overhang residues by comparing the spectra. By comparing the NOESY spectra of ROX labeled and unlabeled DNA, it was found that the presence of ROX resulted in multiple sets of signals and chemical shift changes. The chemical shift difference (∆δ) was calculated between the labeled and unlabeled DNA. For protons that have multiple sets of signals, the difference in every set of spectra were calculated and summed, so that chemical shift changes and multiple signals were both taken into account. The maximum chemical shift differences (∆δmax) among the 5 or 6 assigned protons for every nucleotide were used to represent for the influence of ROX on nucleotide and they were mostly come from H1’ or H2’(H2’’). Results and Discussion General factors influencing fluorescence anisotropy of terminally labeled dsDNA. Fluorescence anisotropy of terminally labeled dsDNA was generally affected by the segmental motion fraction (α) and the harmonic mean correlation time (θH), as described by Eq. 1.1 The harmonic mean correlation time (θH) was an average rotational correlation time used for non-spherical fluorophores such as the rod shaped dsDNA in our case. The depolarization from the fast segmental motion (θF) was much less significant than that from θH,1 thus was negligible in the simplifying process of Eq. 1 (details can be found in the supporting information).

r = (1-α ) r0 / (1 + τ / θ H )

1 where r0 was the fundamental anisotropy of ROX; τ was the lifetime of ROX, which was found to remain roughly constant in our study.1 It is instructive to evaluate the influence of the length of dsDNA, the nature of dyes, and the end structure of DNA to obtain an in-depth understanding of the mechanism of fluorescence anisotropy to facilitate better signal enhancement. The effect of DNA length on fluorescence anisotropy. The rotational diffusion of dsDNA shorter than the persistence length is usually described based on the cylinder model,33-35 thus increasing the length of dsDNA could significantly enlarge the harmonic mean correlation time (θH) for favorable enhancement of the fluorescence anisotropy. The fluorescence anisotropy of dsDNA was basically calculated according to Eq. 1 using the cylinder model (Table S1),35 with detailed calculation provided in the supporting information. Anisotropy values were found to increase dramatically with the length of dsDNA up to 18−20 bp (Figure 1A). However, the fluorescence anisotropy of dsDNA with a longer length leveled off rather than increased steadily as predicted by the cylinder

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model,35 demonstrating that the increased molecular volume did not always effectively contribute to signal enhancement of the fluorescence anisotropy.

intercalated in the molecular frame of the nucleic acid duplex can well report the Brownian motions of the helix. In our study, the terminal labeling was investigated to better serve the analytical purpose, where the dye may bind to nucleotides in a manner different from intercalation. In this case, it seemed that only those nucleotides in proximity of the labeling dye could effectively influence the fluorescence anisotropy. Therefore, the long distance between the far-end nucleotides and dye might be another reason to make the fluorescence anisotropy insensitive to further extended length of dsDNA when the number of base pairs reached up to 18 bp. Such insensitive response to base mismatches provided the feasibility in tuning the free energy change associated with the formation and dissociation of dsDNA, which was highly advantageous for the probe adjustment in detecting different targets with varied binding constants. Yet, the narrow base pair sensitive dynamic range observed here, which could not be expanded by altering the stiffness of dsDNA, illustrated the limited influence of molecular volume on the fluorescence anisotropy of terminally labeled dsDNA. Therefore, alternative enhancement strategy was highly desired.

Figure 1. Predicted and experimental fluorescence anisotropy as a function of the length of dsDNA at 25 °C (A); the effect of GC content (B), the site of single-base mismatch (C) and the number of mismatch (D) on the fluorescence anisotropy with concentrations of 20 nM and 40 nM for fluorescent and complementary strands, respectively.

We tried to expand the base-pair sensitive dynamic range and explain the leveling-off results by altering the temperature and the structural stability of dsDNA. Reducing the temperature only resulted in a globally increased fluorescence anisotropy but did not expand the dynamic range (Figure S1), indicating that motional manners of the dye and dsDNA as well as its influence on fluorescence anisotropy remained unchanged other than that the overall Brownian movement of dsDNA was decelerated. No improved fluorescence anisotropy or expanded length-sensitive dynamic range was observed at the high GC content (Figure 1B). Furthermore, fluorescence anisotropy was found not affected by the site of single-base mismatch (Figure 1C) as well as up to 3-site mismatches (Figure 1D). Decreased fluorescence anisotropy was observed at further increased number of mismatches, specifically 5 consecutive mismatches in the middle of dsDNA and 8 consecutive mismatches at the remote end of the fluorescent dye (Figure 1D). However, dsDNA in such cases could deform from a rigid cylinder, because either the dsDNA was bent into two rigid rods with the mismatched region as the loose linker or the fluorescently labeled dsDNA was literally changed into a structure composed of a substantially shortened double helix and a flexible mismatched moiety which had minimal contribution to the rotational correlation time. Therefore, the effect of minor alteration of the dsDNA structure and the Brownian motion on leveling-off results were both ruled out. In the previous published work,36-42 intercalating dyes, mostly ethidium bromide, was used to establish the effect of DNA internal motions (bending, torsion, and base-pair twisting) on depolarization. Such internal motions of DNA might be a reason for the deviation from rod model prediction. However, in the above mentioned studies, the orientation of the dye

Figure 2. Fluorescence anisotropy of dsDNA (cFAM = 20 nM, cTAMRA = 40 nM, cROX = 20 nM) as a function of pH (A) and the length of dsDNA (B) using different fluorescent dyes.

The effect of the nature of dyes on fluorescence anisotropy. The interaction between the dye and DNA could affect both the molecular volume of dsDNA and the segmental motion of dye, making it promising to improve the fluorescence anisotropy signal via a new approach based on the interaction between the dye and DNA. Considering the electronegative nature of dsDNA, the electrostatic interaction, apart from the van der Waals force,29,43 should be an effective way to confine the segmental motion of dyes. In our study, dsDNA labeled with dyes having the positively charged center (ROX and TAMRA) (Figure S2) exhibited high fluorescence anisotropy signal in a wide pH range (Figure 2A), which was mostly due to the confined segmental motion derived from electrostatic attraction between the dsDNA backbone and dyes. FAM, which had only the negative charged center, suffered from the electrostatic repulsion and exhibited low anisotropy values. Therefore, dyes with the positively charged center were the better choice for fluorescence anisotropy than ones without.28,44 ROX rather than TAMRA was adopted because TAMRA was more susceptible to guanosine base quenching and temperature fluctuation,29,43 leading to unstable signal response (Figure 2B). The effect of end structure of dsDNA on fluorescence anisotropy. The end structure of DNA exerted a significant influence on fluorescence anisotropy of the terminally labeled

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dsDNA via the interaction between the dye and nucleotides. Despite the same level-off phenomenon with similar base pair dynamic range, introducing 6-nt overhang at the opposite side of ROX consistently produced enhanced fluorescence anisotropy for all tested lengths of dsDNA at all three tested temperature levels (Figure S3) , indicating that an appropriate end structure was effective for signal enhancement. As illustrated in Figure 3B, fluorescence anisotropy was found to increase with the length of the C overhang (Figure 3A-b) and reached a plateau at 6 nucleotides for both the 20-nt and the 12-nt F strand, indicating that a 6-nt C overhang was good enough to for signal enhancement, which was superior for experimental design with improved signal.

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well as chemical shift changes (Figure S5 and S6).

Figure 3. Illustration of the terminal structure of fluorescently labeled dsDNA studied (A); the steady-state fluorescence anisotropy as a function of the length of the C overhang (B) and F overhang (C) at the fluorescent terminus. F strand denotes the fluorescently labeled strand and C strand denotes the complementary strand. (cF Strand=50 nM, cC Strand =100 nM.)

In the experimental results based on the Perrin plots (Figure S4, Table S2 and Table S3), no significant difference of the apparent value of r0 (r0 (app)) for dsDNA with blunt end (0.258 ± 0.014) and 6-nt C overhang (0.262 ± 0.017) were observed. The r0 (app) value was mostly affected by the segmental motion fraction (α),1 thus it can be deduced that the α values were similar for both blunt-ended and C overhang structures. This similarity of α values can be attributed to the strong electrostatic interaction between ROX and nucleotides. Without C overhang nucleotides, ROX could already well interact with the electronegative nucleotides at the blunt end, leading to the confinement of segmental motion and a relatively high r0 (app) value (0.258). Therefore, the enhanced fluorescence anisotropy was mostly ascribed to the increased effective rotational volume, rather than improved confinement of segmental motion. As revealed by NMR results summarized in Figure 4, increase of the harmonic mean correlation time could be ascribed to the interaction of ROX with both the double-helical backbone and the C overhang. In 2D 1H-1H NOESY spectra, the proton resonance of some of the bases adjacent to ROX in both the double helix and the C overhang region were significantly affected in the presence of ROX, resulting 2 or 3 sets of signals as

Figure 4. (A) to (D): Illustration of terminally labeled dsDNA used in NMR experiments. All DNA share the same sequence except for abridgment or extension and nucleotides in boxes are those with which the influence of ROX on the chemical shift was considered to be significant. (E) and (F): The maximum chemical shift difference (∆δmax) for each nucleotide, in which the chemical shift difference (∆δ) was defined as the difference of the specific proton between the labeled and unlabeled DNA. For protons that have multiple sets of signals, sum of the differences in every set of spectra were counted. The maximum chemical shift differences (∆δmax) among the 5 or 6 assigned protons for every nucleotide are shown.

The maximum chemical shift difference for every nucleotide between the ROX labeled and unlabeled dsDNA, ∆δmax, was used to evaluate the influence of ROX on DNA nucleotides, and those with chemical shift difference greater than 0.02 were considered to be significantly affected by the presence of ROX. For the blunt-ended structure (Figure 4A), the presence of ROX showed pronounced effect on 4 nucleotide residues

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adjacent to ROX (#1~#4) and 3 residues (#22~#24) complementary to #1~#3 (Figure 4E, S5B), demonstrating the interaction between ROX and dsDNA backbone. In the presence of the 6-nt C overhang (Figure 4B), signals from 2 nucleotides adjacent to ROX (#1~#2) and 2 complementary residues (#23~#24) as well as the first 2 overhang residues (#25~#26) were significantly perturbed in the presence of ROX (Figure 4E, S5A), indicating the interaction between ROX and C overhang in addition to that between ROX and the doublehelical backbone. Furthermore, the other signals from C overhang in the presence of ROX were stronger than that without ROX, suggesting that the C overhang could bind with ROX and was stabilized (Figure S5A). Therefore, the local binding of ROX with C overhang as well as with double-helical backbone resulted in an extended helical axial dimension and possibly a slightly further confined motion of ROX, facilitating a better motional cooperation between ROX and the dsDNA for favorable increase of fluorescence anisotropy. The induced circular dichroism spectra in the range of 540~600 nm confirmed the interaction between ROX and the double-helical structure (Figure S7). Meanwhile, the slower fluorescence anisotropy decay in the presence of 6-nt C overhang compared with blunt-ended 20-bp dsDNA (Figure S8A) confirmed the increased harmonic mean correlation time. As for dsDNA with F overhang structure, fluorescence anisotropy generally decreased with the ascending of F overhang (Figure 3A-c, Figure 3C). Similar to that of the C overhang structure, multiple sets of signals (Figure S5C and S5D) and chemical shift changes were also observed in NOESY spectra due to the pronounced perturbation with the presence of ROX. With a 4-nt F overhang, only two residues in the doublehelical backbone (#19~#20) in addition to all F overhang residues (#1~#4) were significantly affected by ROX (Figure 4D and Figure 4F), suggesting the compromised interaction between ROX and the double-helical backbone. As a result, the formed local complex was not able to incorporate well with the dsDNA backbone to become part of the rigid rotational rod, resulting a reduced rotational correlation time, thus the decreased fluorescence anisotropy. The surprising increase of fluorescence anisotropy for the dsDNA with short 2-nt F overhang could be ascribed to the interaction of ROX with the first couple of nucleotides in the double-helical backbone. As illustrated in Figure 4C and Figure 4F, other than the two residues in F overhang (#1~#2), three base pairs in the double-helical backbone (residues #3~#5 and #20~#22) were significantly affected by ROX, suggesting that ROX could still interact with both the double helix and the overhang nucleotides to extend the helical axial dimension, facilitating an increased θH value and consequently the increased fluorescence anisotropy. When the lengths of double helix and F overhang were both changing at the same time but in opposite directions (Figure 3A-d), a significant descending of fluorescence anisotropy was observed (Figure 3C). We believed such decrease was attributed to the substantially reduced rotational correlation time because the length of the rigid double helix became shorter as F overhang became longer in addition that the flexible overhang nucleotides were not able to be well incorporated with the double helix. Dynamic fluorescence anisotropy of dsDNA with 8-nt F overhang showed faster decay, confirming the pronounced decrease of θH (Figure S8B).

Figure 5. (A) Calculated Kd and LOD as a function of the length of dsDNA with red filled squares showing LOD acquired experimentally and blue open squares showing the calculated LOD; (B) LOD as a function of the concentration of F Strand.

The applications in DNA based recognition. The above studies on the enhancement of fluorescence anisotropy of short dsDNA provided the rationale for improved competitive assay design, in which the signal change associated with target binding only involved the short fluorescent DNA and its hybridized double helix with C overhang structure, assuring the simplicity and generality of the design. The design was demonstrated in this work using DNA detection and aptamer-based detections as examples. DNA detection was taken as an example to better understand the Figure of Merit of the method based on the above design. A direct hybridization mode (Figure 3A-b) was adopted with the target strand as the complementary strand (C strand). The calculated LOD was in good agreement with values obtained experimentally when Kd was at the level about nanomolar or less (Figure 5A, Table S4). Given a concentration of probe DNA (F strand) at 10 nM, the LOD was found to decrease with the length of F strand and level off to about 1~2 nM at 11-nt F strand, and a less than 1 nM LOD was achieved for 15-nt F strand. This design was superior in facilitating a big signal change with target binding because of the distinct anisotropy change between the flexible short F strand and the hybridized dsDNA with an overhang end structure favorable for signal enhancement (Figure S9). The LOD can be further reduced by reducing the concentration of F strand and was verified by the experimental results (Figure 5B).

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Figure 6. Schematic illustration for the target-binding mediated strand-displacement design with signal-on mode (A) and signaloff mode (B). The calibration curves of thrombin (C) and cocaine detection (D).

The competitive manner using strand-displacement reaction was demonstrated for the aptamer-based detection with either a signal-on (Figure 6A) or a signal-off (Figure 6B) mode. Detection of thrombin was used to evaluate the feasibility of the above modes. At the optimized experimental conditions, 10 nM F strand and 7.5 nM aptamer strand, the calibration curve for thrombin presented a linear range up to 15 nM and the LOD of 3.1 nM based on signal-off mode (Figure 6C). Via the signal-on mode, a LOD of 5.1 nM with a 0~40 nM linear range for thrombin was also obtained (Figure 6C). Both modes were feasible with comparable sensitivity, but the signal-off mode had more simplicity than the signal-on mode because less species and equilibria were involved. It is noteworthy that this strand-displacement strategy was also applicable to small molecule detection. So far, small molecules, e.g. cocaine, were usually difficult to detect by fluorescence anisotropy based on direct binding mode. Besides the low binding affinity toward the aptamer, the volume change of the fluorescence species with binding events was negligible, thus much efforts were made to increase the sensitivity of the assay. In the target binding mediated strand-displacement strategy, the fluorescence anisotropy signal change was associated with only initial state and binding state of the F strand, and the volume of the target became much less important in causing the signal change, making detection of small molecules simple and feasible without the help of big DNA-binding protein and nanoparticles. As a demonstration, the signal-off mode was used with overhang configuration for signal enhancement (Figure S10), and a calibration curve was constructed for cocaine with a linear range up to 20 µM and a LOD of 2.2 µM (Figure 6D). Conclusion In summary, fluorescence anisotropy of terminally labeled dsDNA could be improved by tuning the length of dsDNA, choosing dyes with the positively charged center and intelligently designing the end structure of DNA. A simple and universal design rationale was proposed and successfully demonstrated by DNA, protein and even small molecule taking the advantage of C overhang enhancement strategy. The highlights in this study are helpful in experimental designs for facile and efficient enhancement of fluorescence anisotropy signal of fluorescently labeled dsDNA where modification of DNA with nanomaterials or using DNA-binding proteins can be avoided. We hope that this study also helps a more profound understanding of the terminally labeled dsDNA based fluorescence anisotropy, and these insights may further expand the applications of fluorescence anisotropy in the study of thermodynamics and kinetics of DNA based recognition.

ASSOCIATED CONTENT Supporting Information DNA sequences used in experiment, temperature effect, NMR spectra, circular dichroism spectra and dynamic fluorescence anisotropy. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author * Na Li address: College of Chemistry and Molecular Engineering, Peking University, Haidian District, Beijing, 100871, China; Tel: +86-10-62761187; E-mail: [email protected]; Homepage: http: //www.chem.pku.edu.cn/index.php?id=283 * Bin Xia E-mail: [email protected]

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21475004, 21035005 and 21275011).

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Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications.

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Figure 1. Predicted and experimental fluorescence anisotropy as a function of the length of dsDNA at 25 °C (A); the effect of GC content (B), the site of single-base mismatch (C) and the number of mismatch (D) on the fluorescence anisotropy with concentrations of 20 nM and 40 nM for fluorescent and complementary strands, respectively. 90x74mm (300 x 300 DPI)



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Figure 2. Fluorescence anisotropy of dsDNA (cFAM = 20 nM, cTAMRA = 40 nM, cROX = 20 nM) as a function of pH (A) and the length of dsDNA (B) using different fluorescent dyes. 95x41mm (300 x 300 DPI)


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Figure 3. Illustration of the terminal structure of fluorescently la-beled dsDNA studied (A); the steady-state fluorescence anisotropy as a function of the length of the C overhang (B) and F overhang (C) at the fluorescent terminus. F strand denotes the fluorescently labeled strand and C strand denotes the complementary strand. (cF Strand=50 nM, cC Strand =100 nM.) 94x71mm (300 x 300 DPI)



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Figure 4. (A) to (D): Illustration of terminally labeled dsDNA used in NMR experiments. All DNA share the same sequence except for abridgment or extension and nucleotides in boxes are those with which the influence of ROX on the chemical shift was considered to be significant. (E) and (F): The maximum chemical shift differ-ence (∆δmax) for each nucleotide, in which the chemical shift differ-ence (∆δ) was defined as the difference of the specific proton be-tween the labeled and unlabeled DNA. For protons that have multi-ple sets of signals, sum of the differences in every set of spectra were counted. The maximum chemical shift differences (∆δmax) among the 5 or 6 assigned protons for every nucleotide are shown. 89x164mm (300 x 300 DPI)


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Figure 6. Schematic illustration for the target-binding mediated strand-displacement design with signal-on mode (A) and signal-off mode (B). The calibration curves of thrombin (C) and cocaine de-tection (D). 98x88mm (300 x 300 DPI)


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Modulating Fluorescence Anisotropy of Terminally Labeled Double

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