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Fluorescent DNA-protected Silver Nanoclusters for Ligand-HIV RNA Interaction Assay Liang Qi, Yuan Huo, Huan Wang, Jing Zhang, Fu-Quan Dang, and Zhi-Qi Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03166 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015
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Fluorescent DNA-protected Silver Nanoclusters for LigandHIV RNA Interaction Assay Liang Qi,†,‡ Yuan Huo,† Huan Wang,† Jing Zhang,† Fu-Quan Dang,† and Zhi-Qi Zhang*,†,‡ †Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China ‡Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry (Shaanxi Normal University), Ministry of Education, Xi’an 710062, China Supporting Information ABSTRACT: Studying ligand-biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) was used to investigate ligand-RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus transactivation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX-RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand-RNA binding interaction assay.
Interactions between ligands and functional ribonucleic acids (RNAs) have grown in importance for the creation of new drugs that efficiently regulate specific cellular pathways and biological processes since RNA was found to play roles in processes such as transcription and translation.1,2 Studies that have investigated ligand-RNA interactions are relatively fewer in number than those studying other biomacromolecular complexes, and current approaches used to identify proteintargeting small molecules are ill suited for studying RNA binding.3 Some of the main techniques used for analyzing ligandRNA interactions include electrophoretic mobility-shift assays,4 nuclear magnetic resonance (NMR)-based methods,3,5 mass spectrometry (MS)-based methods,6,7 structure-based approaches,8,9 and fluorescence-based approaches.10-12 Electrophoretic mobility-shift assays, ignoring the timeconsuming characteristic, allow for an independent confirmation of binding affinity; NMR-based methods have the ability to precisely determine the binding site of small molecules, but a significant downside is the need to obtain
large quantities of RNA, in some cases isotopically labeled.13 As a label-free technique, MS-based methods can provide ligand-RNA association constants and the stoichiometry of binding. While as ionic strength is known to provide an important contribution to binding energy and selectivity,14 the strength of interaction and binding mode may be different from those observed in an ionic solution-
phase experiment.13 Structure-based approaches are available for the virtual screening for drugs by forecasting the binding mode and affinity between small molecules and RNAs, although it remains necessary to confirm such predictions experimentally. As a simple and general technique, fluorescence-based approaches offer higher sensitivity compared with other approaches (e.g. NMR and MS). Acquired immune deficiency syndrome (AIDS) is caused by an RNA virus, namely human immunodeficiency virus, mainly type I (HIV-1). While some therapeutics, such as HIV1 reverse transcriptase and protease inhibitors, can be effective in controlling AIDS,15-16 additional targets for HIV-1 treatment are urgently needed because of the emergence of drugresistant strains in previously treated and untreated patient populations.17 Two key activators of HIV-1 transcription, namely the trans-activator of transcription (Tat) and regulator of viral expression (Rev) protein can specifically recognize and bind to trans-activation responsive region (TAR) RNA and the Rev response element (RRE) RNA located within the env gene of HIV.18-20 Tat-TAR interactions stimulate the expression of all HIV-1 genes by several orders of magnitude, which is critical for viral replication.21 Rev binding to RRE RNA can regulate viral gene expression by affecting the relative amounts of fully and partially spliced mRNAs that are exported to the cytoplasm.22 Small molecular ligands that bind to the Tat-binding site in TAR RNA or the Rev binding site in RRE RNA can potentially block the formation of Tat-TAR
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and Rev-RRE complexes, thereby inhibiting the functions of the Tat and Rev proteins. Thus, ligand-RNA interactions can potentially play key roles in combating AIDS. Mitoxantrone (MTX) is a common chemotherapeutic agent with proven antitumor activity against breast cancer,23 ovarian cancer,24 and acute leukemia.25 Recently MTX was also shown to bind simultaneously to the Tat-binding site in TAR RNA and the Rev-binding site in RRE RNA through NMR and fluorescent indicator displacement assays, respectively.5,13 Fluorescent silver nanoclusters (AgNCs), especially DNAprotected AgNCs (DNA-AgNCs) have attracted substantial interest.26,27 Key features of AgNCs, such as their ultrasmall size; tunable fluorescence emission; high-luminescence quantum yield; low toxicity; and good photostability, water solubility, and biocompatibility,27 make them very good candidates for fluorescent biosensors and biological applications. Indeed, DNA-AgNCs have been widely applied to detect small metal ions (e.g. Cu2+ and Hg2+) and some biomacromolecules.28-34 However, to our knowledge, DNAAgNCs have not been applied to the study of ligand-RNA interactions. Fluorescent AgNCs biosensors for ligand-RNA interactions should be relatively simple, low-cost and sensitive compared with other reported approaches. In fact, using fluorescent AgNCs for studying ligand-RNA interactions also would eliminate the need for fluorescent indicator or
fluorescently labeled peptides and RNAs generally used in fluorescent ligand displacement-based assays. AgNCs characteristically show low background interference due to their good fluorescent properties. Two quinones (p-benzoquinone and o-naphthoquinone) were previously shown to quench the luminescence of AgNCs.30 MTX possesses an anthraquinone ring, and it was found here that MTX also quenches the fluorescence of AgNCs. However, the fluorescence of AgNCs recovered following MTX disengagement caused by the addition of TAR RNA and RRE RNA to the MTX-AgNCs quenching complex (Figure 1). Because MTX effectively quenched the luminescence of AgNCs and interacted with TAR RNA and RRE RNA, these 3 molecules were selected for a model system to study ligand-RNA interactions using AgNCs as fluorescence probes.
Figure 1. (a) Scheme showing AgNCs as fluorescence probes for MTX-RNA interactions. In the quenched state, the fluorescence of the DNA-protected AgNCs is blocked by MTX binding (grey AgNCs), whereas fluorescence is liberated (yellow AgNCs) following MTX displacement by TAR or RRE RNA (b) The secondary structures of the MTX, TAR RNA and RRE RNA molecules used in this study.
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Experimental section Chemicals and solvents. TAR RNA and RRE RNA were custom-synthesized and HPLC-purified by Takara Biotechnology Co., LTD. (Da Lian, China). A DNA oligonucleotide with the sequence 5′AATTCCCCCCCCCCCCAATT-3′ was synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC)-labeled peptides were synthesized by Shanghai Apeptide Co., Ltd (China). MTX dihydrochloride were purchased from Sigma-Aldrich Co., LLC (USA). All other reagents of analytical grade were commercially available and used without further purification. Tubes and tips were treated with 1% diethyl pyrocarbonate (DEPC; Sigma-Aldrich) prior to use. RNA solutions were all prepared with DEPC water to prevent contamination with ribonucleases. Synthesis of AgNCs. AgNCs protected by DNA with the sequence 5′-AATTCCCCCCCCCCCCAATT-3′ were typically prepared27 using concentrations of DNA, BH4 ions, and Ag ions of 25 µM, 150 µM and 150 µM, respectively. Briefly, 100 µL of DNA (100 µM) was mixed with 270 µL of PBS (20 mM, pH 7.0), after which 10 µL of an aqueous AgNO3 solution (6 mM) was added, followed by vigorous shaking for 10 min. Twenty microliters of freshly prepared aqueous NaBH4 solution (3 mM) was added to the above solution, followed by vigorous shaking for another 30 s. The solutions were kept in the dark and allowed to react for 4 h. The absorption and emission wavelengths of the fluorescent AgNCs were then measured. Fluorescence measurements. All fluorescence spectra measurements were obtained with an LS 55 Fluorescence Spectrometer, using a quartz cuvette with a 0.2-cm path length. The emission spectra of AgNCs were recorded from 420 to 700 nm. Both the emission and excitation slits were set to 5 nm, the scanning speed was 120 nm min-1 and the photomultiplier voltage was set to auto. In fluorescence-quenching experiments, fluorescence spectra were measured in binding assays after adding a variable amount of MTX to a fixed concentration of AgNCs. MTX and AgNCs were mixed thoroughly and incubated at room temperature for 5 min in 20-mM phosphate buffer saline (PBS; pH 7.0, containing 20 mM NaCl) before testing, unless otherwise specified. All fluorescence spectra were measured at least in triplicate. In fluorescence-recovery experiments, fluorescence spectra were measured after adding a variable amount of TAR or RRE RNA to a fixed concentration of the MTX-AgNCs complex. The resulting solutions were incubated at room temperature for 10 min in 20-mM PBS before testing, unless otherwise specified. All fluorescencespectra were measured at least 3 times. Fluorescence polarization analysis was performed using excitation and emission wavelengths of 495 and 517 nm, respectively, with a slit bandwidth of 5 nm. The integration time was 1 s. Fluorescence polarization was measured at 517 nm for quantitative analysis. Circular dichroism (CD) measurements. CD spectra were measured using a ChirascanTM CD Spectrometer (Applied Photophysics Ltd. Co., United Kingdom) and a quartz cuvette with a 0.2-cm path length. The scanning speed was set at 200 nm•min-1, and the optical path was set at 1 mm. The final
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Analytical Chemistry
spectra were obtained after background correction (air and PBS buffer). CD spectra in binding assays were measured by keeping the concentration of AgNCs constant while adding a variable amount of MTX. The MTX-AgNCs complexes were mixed thoroughly and incubated for 10 min in 20-mM PBS before testing. All CD spectra were scanned at least 3 times. Electron microscopy characterization. Samples were prepared on carbon-film-covered copper grids, and images were obtained on a JEOL JEM-2100 Transmission Electron Microscope (TEM) at 200 KV. Mass spectrometry. Electrospray ionization (ESI)-MS was performed with a maxisTM UHR-TOF Mass Spectrometer (Bruker, Leipzig, Germany). The ionization mode was negative. The MS parameters were: scan range m/z = 50–2500; nebulizer flow, 0.3 bar; dry-gas flow rate, 3.0 L•min-1; dry-gas temperature, 120ºC; capillary voltage, 4000 V; end-plate offset voltage, −500 V; collision cell RF, 1500.0 Vpp.
Results and discussion Fluorescence quenching of AgNCs following the addition of MTX. The fluorescence spectra of AgNCs before and after the addition with MTX were measured. Following their synthesis, 15 µL of AgNCs was mixed with MTX at various concentrations, and the final volume was adjusted to 200 µL with 20-mM phosphate buffer solution (pH 7.0, containing 20 mM NaCl). AgNCs exhibited significant fluorescence emission at 603 nm in the absence of MTX. However, AgNCs fluorescence was quenched by MTX in a concentrationdependent manner, with ~95% fluorescence quenching observed in the presence of 5000 nM MTX (Figure 2). The decrease of fluorescence intensity was linear when the MTX concentration was plotted on a logarithmic scale (Figure S1 in the Supporting Information).
Figure 2. Concentration-dependent inhibition of AgNCs fluorescence by the addition of MTX (50, 500, 1250, 2500 and 5000 nM) in phosphate buffer solution. The quenching constant (Ksv) was calculated using the following equation, as described previously.35
∆
(1)
where ∆F is the difference in fluorescence measured in the absence (F0) and presence (F) of MTX at concentration [Q], fa is the fraction of maximum accessible fluorescence, and Ksv is
the effective quenching constant for the accessible fluorophores. The dependence of F0/∆F on the reciprocal value of the MTX concentration ([Q]-1) is linear with the slope equaling the value of (faKsv)-1. The value fa-1 is the Y-intercept. The constant Ksv is a quotient of the ordinate fa-1 and the slope (faKsv)-1. The quenching rate constant (Kq) can be determined as Kq=Ksv/το. Curve fitting analysis for eq. (1) suggested that the fluorescence of AgNCs could be quenched by MTX with a quenching constant (Ksv) of 1.45×106 M-1 (Figure S2). It is well known that there are 2 types of quenching processes: static and dynamic quenching.34 The value of Kq (~1.4×1012 L s-1mol-1) was far greater than the maximum diffusion
collision quenching rate constant of various quenchers with biopolymers (2.0×1010 L s-1mol-1),37 indicating the fluorescence quenching of AgNCs caused by MTX occurs as a static quenching process. TEM was previously used to study the mechanism of fluorescence enhancement of AgNCs upon the addition of ATP.33 In this study, TEM was used to confirm that MTX quenches AgNCs fluorescence. A change in AgNCs size after MTX addition was observed. TEM images indicated that the average size of AgNCs was ~4 nm (Figure 3a), which increased to ~11 nm in the presence of MTX (Figure 3b), indicating that the addition of MTX induced the aggregation of AgNCs. Because the fluorescence intensity of AgNCs decreases as AgNCs size increases, thus the increase of AgNCs size after addition of MTX was the main contributor to the fluorescence quenching of AgNCs. The UV-vis absorption spectra of AgNCs and MTX were measured and MTX showed clear absorption at around 610 nm and 670 nm, which overlapped with the absorption and emission spectra of AgNCs at the experimental concentration ratio used (Figure S3). Thus, the inner-filter effect of fluorescence was considered as another reason for fluorescence quenching. In addition, CD experiments were also conducted to measure conformational changes of DNA occurring in the presence or absence of MTX. The CD spectra showed that MTX addition caused no obvious structural changes of template DNA (Figure S4), suggesting that MTX did not quench the AgNCs fluorescence by changing the conformation of DNA.
Figure 3. TEM images of AgNCs (a) in the absence and (b) presence of MTX. MTX-RNA interaction analysis. A fluorescence recovery experiment was conducted to study ligand-RNA interactions, using AgNCs as fluorescence probes. An MTX concentration of 2.5 µM, which corresponded to ~85% AgNCs quenching, was chosen for the recovery experiments, and TAR or RRE RNA was added to the AgNCs-MTX quenching system to study their effects on fluorescence. The measured fluorescence
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increased linely with increasing concentrations of TAR RNA, eventually reaching ~82% of that of free AgNCs in the presence of 5 µM TAR RNA (Figure 4a). Similar results were obtained with RRE RNA, with the measured fluorescence reaching ~90% of that of free AgNCs with 1.5 µM RRE RNA (Figure 4b).
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Assuming that [Df]/[Dt]= F0/F, it follows that, 'R D( D) * D D) +1 * R R ) *
.
D)
-
(7) (8)
Based on eq. (7), (8) and the assumption [Df]/[Dt]= F0/F, the following relationship was found， /. 0
!=
/. 0 1 #2 .3 4+ $2 -5
!
(9)
Eq. (9) follows that,
log
Figure 4. Fluorescence spectra of free AgNCs and concentration-dependent increases in fluorescence after adding (a) TAR RNA (50, 200, 500, 1000 and 5000 nM) and (b) RRE RNA (25, 100, 300, 500, 800 and 1500 nM) to the MTXAgNCs complexes. Identical and independent binding sites in the RNA (R) and a single binding site for the drug (D; i.e. MTX) were assumed in the present study, and the binding affinity of RNA at each binding site was assumed to be equal. Based on these assumptions, the following equations describing interactions between D and R were derived,
R + D RD
(2)
R D R D
(3)
R D R D
(4)
R D R D
(5)
Ka can be written as,
!
"# $% !
#& ! $&
=
(6)
where [Rf] and [Df] are the concentrations of free RNA and MTX, respectively. [R1/nD] is the concentration of the newly formed complex R1/nD.
.
log 9 log + R ) *
.
D) -
(10)
where [Rt] and [Dt] represent the total concentrations of RNA and MTX, respectively. Plotting log (F0-F)/F versus log ([Rt] (1/n) × [Dt] × (F0-F)/F0) showed a linear fit, which agreed with the assumption that 1/n in the bracket equaled 1. Thus, the slope of 1/n could be obtained. If the slope obtained was not equal to 1/n, then it was substituted into the bracket, and the curve was redrawn in an iterative process until the value for 1/n inside the bracket was equal to the slope of 1/n outside the bracket. This process enabled determination of the number of binding sites (n), after which the binding constant (Ka) of RNA to the MTX-AgNCs complex could be obtained as the Yintercept. These deductions were based on a previous description.38 Eq. (10) was finally curve fitted as y= 0.2937 x + 2.0935 (R2=0.9926) for TAR RNA and y = 0.2999x + 2.2849 (R2=0.9954) for RRE RNA via the above calculation processes. MTX was predicted to interact with RNA with a relative binding constant (Ka) of ~1.34×107 M-1 with 3.4 binding sites for TAR RNA (Figure S5), which is consistent with the reported dissociation constant (Kd= 0.055 µM),5 and a Ka of ~4.13×107 M-1 and with 3.3 binding sites and for RRE RNA (Figure S6). In addition, control experiments were conducted to verify the reliability of these theoretical results. The fluorescence spectra of AgNCs and MTX were measured separately in the absence and presence of either RNA, and the results showed that AgNCs fluorescence was slightly quenched after mixing with TAR RNA and RRE RNA. The major contribution toward fluorescence quenching potentially originated from electrostatic interactions between the positively charged base residues of RNA and the negatively charged AgNCs (Figure S7), while the fluorescence of MTX was not enhanced after the addition of either RNA (Figure S8). These results may help explain why the fluorescence recovery rate did not reach 100%, and why disengagement of AgNCs-MTX complexes by TAR or RRE RNA led to recovered fluorescence. Confirmation of MTX binding to TAR RNA and RRE RNA. Fluorescence polarization (FA) is an attractive method for studying intermolecular interactions39-41 and was applied in this study to confirm the interactions between MTX with TAR and RRE RNA. The fluorescence polarization value (1 P=1000 mP) was calculated using the following equation, m; 1000
=
.>= ? =
@>= ?
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Where Ivv and Ivh are the fluorescence intensity horizontal to and vertical to the excitation plane, respectively, and G=Ihv/Ihh. FITC-Rev could interact with RRE RNA to increase its polarization, and FITC-Tat bound TAR RNA to form a complex with high polarization. Various concentrations of MTX were added to the pre-formed complexes. As the concentration of MTX increased, polarization of the analytes initially increased, then decreased to the polarization of the free FITC-peptide (Figure 5). It can be concluded that MTX preferentially bound to other regions rather than the peptide binding sites of RNAs at low concentrations, which made the molecular weight of the whole complex increase, leading to decreased rotation, and finally causing the increased polarization. Such a change of polarization indicated that ternary complex formation was possible at low MTX concentrations, implying the existence of multiple MTXbinding sites on RNAs, which is consistent with the above theoretical calculations. As the MTX concentrations increased, it interacted with RNA at the peptide binding sites, until FITCRev and FITC-Tat were both almost completely displaced with the addition of higher concentrations of MTX, as the observed reductions in polarization.
Figure 6. Negative-ion ESI mass spectra for the titration of MTX (100 µM concentration) with (a) TAR RNA and (b) RRE RNA (20 µM final concentrations). Figure 5. Fluorescence polarization of FITC-Tat after adding (a) various concentrations of MTX (0, 125, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 nM) to the FITC-Tat (50 nM) and TAR RNA (50 nM) complexes and (b) MTX (0, 125, 375, 625, 875, 1250, 2500, 3750, 5000 and 6250 nM) to the FITCRev (50 nM) and RRE RNA (50 nM) complexes. MS was used to confirm the binding of MTX to TAR and RRE RNA. TAR RNA (13 negative charges) and RRE RNA (14 negative charges) were studied in negative-ion mode, and the binding of multiple MTX molecules occurred at high MTX concentrations. MTX-TAR RNA complexes carrying 9-13 negative charges and MTX-RRE RNA complexes carrying 1014 negative charges were observed, and the maximum binding stoichiometries of MTX binding to TAR RNA and RRE RNA were 4 and 3, respectively (Figure 6), which was consistent with the above theoretical calculations.
Conclusions In summary, DNA-protected AgNCs was used to study the interactions between MTX with TAR RNA and RRE RNA in this study. In addition, the binding constants and number of binding sites for MTX to TAR and RRE RNA were determined. FA and MS results were used to confirm MTX binding to TAR and RRE RNA, and the results were consistent with theoretical calculations performed in this study. The quenching constant and mechanism of MTX quenching of AgNCs fluorescence were also explored. The interactions between other ligands quenching AgNCs fluorescence with TAR and RRE RNA could reference the mode of MTXAgNCs. However, if the ligands could not quench AgNCs fluorescence, ligands-RNA interactions could still be investigated through the displacement of MTX. AgNCs could also be used as fluorescence probes for characterizing other ligand-RNA binding interaction assays, and the deduced formulae are also appropriate for the determining of binding constants and number of binding sites for other complexes. This strategy further support nanotechnology applications in
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the area of ligand-RNA interaction assays, since other representative fluorescent nano particles can also be used as fluorescence probes for ligand-RNA interactions.
ASSOCIATED CONTENT Supporting Information Curve fitting analysis of fluorescence intensity of AgNCs with logarithm concentration of MTX; Curve fitted analysis of eq. (1) for MTX quenching the fluorescence of AgNCs; Excitation and emission spectra of AgNCs and absorption spectra of AgNCs and MTX. CD spectra of AgNCs in the absence and presence of MTX; Curve fitted analysis of eq. (10) for MTX binding to TAR RNA; Curve fitted analysis of eq. (10) for MTX binding to RRE RNA; Fluorescence of AgNCs in the absence and presence of RRE RNA and TAR RNA Fluorescence of MTX in the absence and presence of TAR RNA and RRE RNA. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *
[email protected] Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work is financially supported by National Natural Science Foundation of China (No. 21275098), Natural Science Grand Research Program of Shaanxi Province (No. 2013SZS08-Z01), Doctor Base Foundation of Chinese Ministry of Education (No. 20110202110005) and Innovation Funds of Graduate Programs of Shaanxi Normal University (No. 2013CXS048).
REFERENCES (1) Tian, R.; Xu, S.; Lei, X.; Jin, W.; Ye, M.; Zou, H. Trac-Trend. Anal. Chem. 2005, 24, 810-825. (2) Cooper, T. A.; Wan, L.; Dreyfuss, G. Cell 2009, 136, 777-793. (3) Stelzer, A. C.; Frank, A. T.; Kratz, J. D.; Swanson, M. D.; Gonzalez-Hernandez, M. J.; Lee, J.; Andricioaei, I.; Markovitz, D. M.; Al-Hashimi, H. M. Nat Chem Biol 2011, 7, 553-559. (4) Sczepanski, J. T.; Joyce, G. F. J. Am. Chem. Soc. 2013, 135, 13290-13293. (5) Lee, J.; Vogt, C. E.; McBrairty, M.; Al-Hashimi, H. M. Anal. Chem. 2013, 85, 9692-9698. (6) Sannes-Lowery, K. A.; Hu, P.; Mack, D. P.; Mei, H. Y.; Loo, J. A. Anal. Chem. 1997, 69, 5130-5135. (7) Hofstadler, S. A.; Sannes-Lowery, K. A. Nature reviews. Drug discovery 2006, 5, 585-595. (8) Lind, K. E.; Du, Z.; Fujinaga, K.; Peterlin, B. M.; James, T. L. Chem. Biology 2002, 9, 185-193. (9) Hermann, T. Current opinion in structural biology 2005, 15, 355366. (10) Parsons, J.; Castaldi, M. P.; Dutta, S.; Dibrov, S. M.; Wyles, D. L.; Hermann, T. Nat. Chem. Biol. 2009, 5, 823-825. (11) Zhang, J. H.; Umemoto, S.; Nakatani, K. J Am Chem Soc 2010, 132, 3660-3661. (12) Umemoto, S.; Im, S.; Zhang, J. H.; Hagihara, M.; Murata, A.; Harada, Y.; Fukuzumi, T.; Wazaki, T.; Sasaoka, S.; Nakatani, K. Chem-Eur. J. 2012, 18, 9999-10008
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(16) Cohen, J. Science 1996, 272, 195. (17) Hirsch, M. S.; Brun-Vezinet, F.; Clotet, B.; Conway, B.; Kuritzkes, D. R.; D'Aquila, R. T.; Demeter, L. M.; Hammer, S. M.; Johnson, V. A.; Loveday, C.; Mellors, J. W.; Jacobsen, D. M.; Richman, D. D. Clin. Infect. Dis. 2003, 37, 113-128. (18) Bagashev, A.; Sawaya, B. E. Virol. J 2013, 10, 358. (19) Olsen, H. S.; Cochrane, A. W.; Dillon, P. J.; Nalin, C. M.; Rosen, C. A. Gene. Dev. 1990, 4, 1357-1364. (20) Zapp, M. L.; Hope, T. J.; Parslow, T. G.; Green, M. R. P. Natl. Acad. Sci. USA 1991, 88, 7734-7738. (21) Karn, J. J Mol. Biol. 1999, 293, 235-254. (22) Battiste, J. L.; Mao, H.; Rao, N. S.; Tan, R.; Muhandiram, D. R.; Kay, L. E.; Frankel, A. D.; Williamson, J. R. Science 1996, 273, 1547-1551. (23) Neidhart, J. A.; Gochnour, D.; Roach, R.; Hoth, D.; Young, D. J. Clin. Oncol. 1986, 4, 672-677. (24) Lawton, F.; Blackledge, G.; Mould, J.; Latief, T.; Watson, R.; Chetiyawardana, A. D. Cancer Treat. Rep. 1987, 71, 627-629. (25) Brito-Babapulle, F.; Catovsky, D.; Slocombe, G.; Newland, A. C.; Marcus, R. E.; Goldman, J. M.; Galton, D. A. Cancer Treat. Rep. 1987, 71, 161-163. (26) Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M. J. Am. Chem. Soc. 2004, 126, 5207-5212. (27) Richards, C. I.; Choi, S.; Hsiang, J. C.; Antoku, Y.; Vosch, T.; Bongiorno, A.; Tzeng, Y. L.; Dickson, R. M. J. Am. Chem. Soc. 2008, 130, 5038-5039. (28) Lan, G. Y.; Huang, C. C.; Chang, H. T. Chem. Commun. 2010, 46, 1257-1259. (29) Huang, Z. Z.; Pu, F.; Hu, D.; Wang, C. Y.; Ren, J. S.; Qu, X. G. Chem-Eur. J. 2011, 17, 3774-3780. (30) Liu, X. Q.; Wang, F.; Niazov-Elkan, A.; Guo, W. W.; Willner, I. Nano Lett 2013, 13, 309-314. (31) Yeh, H. C.; Sharma, J.; Han, J. J.; Martinez, J. S.; Werner, J. H. Nano Lett. 2010, 10, 3106-3110. (32) Liu, X.; Wang, F.; Aizen, R.; Yehezkeli, O.; Willner, I. J. Am. Chem. Soc. 2013, 135, 11832-11839. (33) Shen, C.; Xia, X.; Hu, S.; Yang, M.; Wang, J. Anal. Chem. 2015, 87, 693-698. (34) Petty, J. T.; Sergev, O. O.; Kantor, A. G.; Rankine, I. J.; Ganguly, M.; David, F. D.; Wheeler, S. K.; Wheeler, J. F. Anal. Chem. 2015, 87, 5302-5309. (35) Hu, Y. J.; Liu, Y.; Xiao, X. H. Biomacromolecules 2009, 10, 517-521. (36) Lakowitcz, J. R.; Plenum Press, New York, 1999, pp 237-259. (37) Ware, W. R. J. Phys. Chem. 1962, 66, 66455-66458. (38) Bi, S.; Qiao, C.; Song, D.; Tian, Y.; Gao, D.; Sun, Y.; Zhang, H. Sensor. Actuat. B: Chem.2006, 119, 199-208. (39) Wojtuszewski, K.; Mukerji, I. Biochemistry 2003, 42, 3096-3104. (40) Souza-Fagundes, E. M.; Frank, A. O.; Feldkamp, M. D.; Dorset, D. C.; Chazin, W. J.; Rossanese, O. W.; Olejniczak, E. T.; Fesik, S. W. Anal. Biochem. 2012, 421, 742-749. (41) Choi, J. W.; Kang, D. K.; Park, H.; DeMello, A. J.; Chang, S. I. Anal. Chem. 2012, 84, 3849-3854.
(13) Thomas, J. R.; Hergenrother, P. J. Chem. Rev. 2008, 108, 1172-1224. (14) Pilch, D. S.; Kaul, M.; Barbieri, C. M.; Kerrigan, J. E. Biopolymers 2003, 70, 58-79.
(15) Davies, D. R. Ann. Rev. Biophys. Biophys. Chem. 1990, 19, 189215.
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