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Oligonucleotide-Based Fluorescent Probe for Sensing of Cyclic Diadenylate Monophosphate in Bacteria and Diadenosine Polyphosphates in Human Tear Jia-Hui Lin, Wei-Bin Tseng, Kai-Cheng Lin, Chih-Yi Lee, Shanmugam Chandirasekar, Wei-Lung Tseng, and Ming-Mu Hsieh ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00425 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 5, 2016
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Oligonucleotide-Based Fluorescent Probe for Sensing of Cyclic Diadenylate Monophosphate in Bacteria and Diadenosine Polyphosphates in Human Tear
Jia-Hui Lin,a Wei-Bin Tseng,b Kai-Cheng Lin,c Chih-Yi Lee,a Shanmugam Chandirasekar,a Wei-Lung Tseng*a,d, and Ming-Mu Hsieh*b
a
Department of Chemistry, National Sun Yat-sen University, Taiwan
b
Department of Chemistry, National Kaohsiung Normal University, Taiwan
c
Department of Orthopaedics, Kaohsiung Veterans General Hospital, Taiwan
d
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan
Correspondence: * Prof. Dr. Wei-Lung Tseng, Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan 804. E-mail:
[email protected] Fax: 011-886-7-3684046. * Prof. Dr. Ming-Mu Hsieh, Department of Chemistry, National Kaohsiung Normal University, No.62, Shenjhong Rd., Yanchao District, Kaohsiung City, Taiwan 82446. 1 ACS Paragon Plus Environment
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E-mail:
[email protected] Phone: 011-886-7-7172930 ext. 7121 J.-H. Lin and W.-B. Tseng contributed equally to this work.
ABSTRACT Cyclic
diadenylate
monophosphate
(c-di-AMP)
and
P1,P5-diadenosine-5'
pentaphosphate (Ap5A) have been determined to play important roles in bacterial physiological processes and human metabolism, respectively. However, few, if any, methods have been developed that use fluorescent sensors to sense c-di-AMP and Ap5A in the real world. To address this challenge, this study presents a fast, convenient, selective, and sensitive assay for quantifying c-di-AMP and Ap5A fluorescence based on the competitive binding of diadenosine nucleotides and a polyadenosine probe to coralyne. The designed probe consists of a 20-mer adenosine base (A20), a fluorophore unit at the 5’-end, and a quencher unit at the 3’-end. Through A2coralyneA2 coordination, coralyne causes a change in the conformation of A20 from that of a random coil to a folded structure, thus enabling the fluorophore to be close to the quencher. As a result, fluorescence quenching occurs between the two organic dyes. When the A20·coralyne probe encounters the diadenosine 2 ACS Paragon Plus Environment
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nucleotide, the resulting complex of coralyne and diadenosine nucleotides forces the removal of coralyne from the probe. Such a conformational change in the probe leads to the restoration of fluorescence. Within a short analysis time of 1 min, the proposed probe provides high selectivity toward c-di-AMP and Ap5A over other common nucleotides. The probe’s detection limit at a signal-to-noise ratio of 3 for both c-di-AMP and Ap5A were estimated to be 0.4 and 4 μM, respectively. The practicality of the proposed probe was demonstrated by quantifying c-di-AMP in bacteria lysates and Ap5A in human tears.
Keywords: cyclic diadenylate monophosphate; diadenosine polyphosphate; sensor; adenosine; fluorescence
Diadenosine nucleotides are classified into two types, including diadenosine polyphosphates and cyclic diadenosine monophosphate (c-di-AMP). Diadenosine polyphosphates are found ubiquitously in tissue lysates and biological fluids (blood plasma, tears, and urine),1 while c-di-AMP has recently been recognized as an important second-messenger nucleotide in bacteria.2 Diadenosine polyphosphates in biological fluids have been demonstrated to be involved in the control of vascular tone, the growth of vascular smooth muscle cells, cardio activity, and platelet aggregation.3-4 3 ACS Paragon Plus Environment
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In addition, c-di-AMP has been linked with bacterial growth, cell-wall homeostasis, biofilm formation, potassium uptake, and cellular resistance to antibiotics.5 Considering the important roles of diadenosine polyphosphates in neurotransmission and c-di-AMP in signaling pathways, a rapid, convenient, selective, and sensitive method is highly desirable for quantifying diadenosine polyphosphate and c-di-AMP in complex matrices. Analytical techniques such as high-performance liquid chromatography (HPLC) combined with mass spectrometry,6 HPLC coupled with UV absorption,7 and tandem boronate affinity-ion exchange chromatography combined with UV absorption8 have been well established for quantifying diadenosine polyphosphates in biological fluids with high sensitivity. Compared to those for diadenosine polyphosphates, relatively few methods are available for identifying c-di-AMP.9-10 For example, a competitive enzyme-linked immunosorbent assay for sensing c-di-AMP was developed based on the competition between biotin-labeled c-di-AMP and c-di-AMP when binding to a c-di-AMP binding protein.9 The limitation of this method is that it requires a long analysis time and involves a complicated procedure. Moreover, halide anion-induced fluorescence quenching of coralyne was implemented to detect the level of c-di-AMP, diadenyl cyclase-induced synthesis of c-di-AMP, phosphodiestearses YybT-triggered degradation of c-di-AMP, and the inhibitor of diadenyl cyclase.10-11 In this assay, halide 4 ACS Paragon Plus Environment
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anion such as bromide and iodide was found to be efficient to quench the fluorescence of coralyne. Because coralyne can complex with more than six c-di-AMP molecules through the coordination between diadenosine moieties and coralyne, the presence of c-di-AMP restored the fluorescence of coralyne in the presence of halide anion. However, coralyne as a fluorescent probe suffers from small fluorescent turn-on ratios and excitation/emission wavelength in visible-light region. RNA-based fluorescent sensors were recently developed for fluorescent turn-on detection of c-di-AMP levels in live bacterial cells.12 This probe provided the lowest detectable concentration (50 nM) for c-di-AMP as compared to the previously reported methods. The limitation of this probe is the difficulty in designing its stem lengths and sequences. Oligonucleotide-based fluorescent sensors have been intensively utilized for fluorescence turn-on/off detection of a wide variety of analytes such as nucleic acids, proteins, small molecules, and metal ions because of their advantages of simplicity, rapidity, sensitivity, diversity, and selectivity.13-22 Typically, a fluorophore- and quencher-modified oligonucleotide probe consists of a predefined base sequence and length, and is designed to recognize specific target analytes.23 Nevertheless, as yet, no study has explored the development of an oligonucleotide-based fluorescent sensor for probing diadenosine polyphosphates and c-di-AMP. Recently, adenosine-rich oligonucleotides were applied as an effective fluorescent platform for the sensitive and 5 ACS Paragon Plus Environment
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selective detection of nucleic acids,24 heparin,25 coralyne,26 and oversulfated chondroitin sulfate27 based on the analyte-induced removal of coralyne from an adenosine-rich oligonucleotide. Coralyne promotes a stable antiparallel duplex of polydeoxyadenosine with a stoichiometry of 1 coralyne per 4 adenine bases. 28 Also, Sintim’s group has demonstrated that coralyne binds to c-di-AMP with a stoichiometry greater
than
1:6.10
Motivated
by
these
results,
we
have
developed
a
polyadenosine-based fluorescent probe for the rapid, sensitive, and selective detection of c-di-AMP and diadenosine polyphosphates. Instead of halide anion-induced fluorescence quenching of coralyne and c-di-AMP-triggered increase in the fluorescence of coralyne used in the previous study,10 polyadesnone labeled with fluorophore at the 5′-end and a quencher at the 3′-end was used to probe c-di-AMP based on the competitive binding of diadenosine nucleotides and polyadenosine to coralyne. This study successfully applied this probe for determining c-di-AMP in bacteria and P1,P5-diadenosine-5' pentaphosphate (Ap5A) in human tears. Compared to the
previously
reported
sensors
for
detecting
coralyne,9-10
our
designed
oligonucleotide-based probe has high fluorescence turn-on ratios, tunable excitation and emission wavelengths, fast response time, and/or simple detection procedure.
EXPERIMENTAL SECTION 6 ACS Paragon Plus Environment
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Chemicals.
Ap5A,
diadenosine
triphosphate,
diadenosine
tetraphosphate,
adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thymidine triphosphate,
cytidine
triphosphate,
guanosine
monophosphate,
guanosine
triphosphate, hydrochloric acid (HCl), sodium chloride (NaCl), H3PO4, NaH2PO4, Na2HPO4, and Na3PO4 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cyclic diguanylate monophosphate (c-di-GMP) and c-di-AMP were ordered from InvivoGen (San Diego, California, USA). All DNA samples were ordered from Neogene Biomedicals Corporation (Taipei, Taiwan). Milli-Q ultrapure water (Hamburg, Germany) was used in all of the experiments. Apparatus. Absorption and Fluorescence spectra were collected on Hitachi F-7000 fluorometer (Hitachi, Tokyo, Japan). Fluorescence polarization were recorded with a Hitachi F-7000 fluorometer coupled to an auto-polarization measurement. Circular dichroism (CD) was measured using a JASCO model J-810 CD spectropolarimeter (JASCO, Tokyo, Japan). Sample Preparation. All DNA samples and analytes were prepared in a solution containing 50 mM phosphate (pH 4.58.5) and NaCl (0100 mM). The DNA probes (100 nM, 50 μL) reacted with coralyne (5 μM, 50 μL) at ambient temperature for 3 min. c-di-AMP (025 μM, 400 μL) and Ap5A (0125 M) were separately mixed with the resulting solution (100 μL) at ambient temperature. After 020 min, all 7 ACS Paragon Plus Environment
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mixture were transferred separately into a series of 1-mL cuvette. Their fluorescence spectra were measured with excitation wavelength set at 480 nm. To test the selectivity of the proposed probe, this study replaced c-di-AMP with other nucleotides. Determination of c-di-AMP in Bacteria. Streptococcus aureus (BCRC 15211; S. aureus) was obtained from the Bioresource Collection and Research Center (BCRC) in Taiwan. Samples of S. aureus were grown in Luria-Bertani broth (25 g/L; LB-N; Difco, Becton Dickinson, Sparks, Md.) at 37°C for 24 h. When the optical density of S. aureus at 600 nm (OD600nm) were cultured to 2.65, they were centrifuged at 12000 rpm for 15 min; 1.00 OD600nm corresponds to approximately 5 1081 109 cells/mL. The bacterial pellet (50 μL) was re-suspended in a solution of Tris-HCl (950 μL, 50 mM; pH 8.0) and then treated by ultrasonic cell disruptor (Branson Digital Sonifier model 450, USA) at 20 KHz for 10 min. To remove bacterial debris, the lysate (1 mL) were centrifuged at 12000 rpm for 15 min. The obtained supernatant (450 μL) was spiked with standard solutions (50 L) of c-di-AMP at concentrations of 0, 5, 10, 20, 25, and 50 μM. The spiked samples (400 μL) were incubated with a solution (100 μL) containing 50 mM phosphate (pH 7.5), 10 nM cyanine 3- and BHQ2-labeled oligonucleotide probe, and 0.5 M coralyne at ambient temperature for 1 min. Their fluorescence spectra were collected at an excitation wavelength of 540 nm. 8 ACS Paragon Plus Environment
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Determination of Ap5A in human tear. Human tear samples were collected from a healthy adult female through capillary tube method. The samples (390 L) were spiked with various concentrations (05 mM, 10 L) of standard Ap5A. We equilibrated the spiked samples (400 μL) with a solution (100 μL) consisting of 50 mM phosphate (pH 7.5), 10 nM MB, and 0.5 M coralyne at ambient temperature for 1 min and recorded their fluorescence spectra at an excitation wavelength of 480 nm.
Results and Discussion Sensing of c-di-AMP. Figure 1 illustrates how fluorophore and quencher-labeled polyadenosine is used to sense diadenosine nucleotides. The designed probe is composed of 20-repeat adenosines (A20), a reporter of carboxyfluorescein (FAM) at the 5′-end, and a quencher of BHQ1 at the 3′-end. A random coil of the designed probe fluoresces weakly because the FAM is far away from the BHQ1. The presence of coralyne
drives
the
coordination
of
adensoine2coralyneadenosine2
(A2coralyneA2), thus enabling the designed probe to convert from a random coil conformation into a folded structure. Because of the spatial proximity between FAM and BHQ1, the fluorescence of the FAM is quenched by the BHQ1. The addition of c-di-AMP to the A20·coralyne probe triggers the removal of coralyne through stacking and hydrophobic interaction,10 thereby restoring the fluorescence of the FAM. 9 ACS Paragon Plus Environment
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Because Ap5A contains two adenosine moieties, it also exhibits the ability to switch on the fluorescence of the A20·coralyne probe. To test the feasibility of our proposed approach, coralyne was used to conduct fluorescence quenching in FAM- and BHQ1-modified A20. Because the complex of A2coralyneA2 is quite stable at a neutral pH, the coordination of FAM- and BHQ1-modified A20 and coralyne was performed in a 50-mM phosphate buffer (pH 7.5). As the concentration of coralyne increased, the fluorescence of the FAM at 520 nm gradually decreased within the range of 5 nM to 5 M and leveled off to a constant value above 0.5 M (Figure 2A). Apparently, a stable complex of coralyne and A20 forms at concentrations of coralyne greater than 0.5 μM. Thus, the A20·coralyne probe was prepared by incubating 0.5 μM coralyne with 10 nM A20. Figure 2B reveals the time-dependent fluorescence intensity of the A20·coralyne probe (10 nM) at 520 nm after the addition of 5 μM c-di-AMP. The fluorescence of the A20·coralyne probe intensified with time and reached a saturation level after 1 min. Figure 2C shows the fluorescence spectra of the A20·coralyne probe in the presence of the increased concentration of c-di-AMP at a fixed 1-min time interval. As the concentration of c-di-AMP increased, the fluorescence of the A20·coralyne probe and the (IF IF0)/IF0 value were gradually enhanced (inset in Figure 2C). IF0 and IF correspond to the fluorescence intensity of the A20·coralyne probe at 520 nm in the 10 ACS Paragon Plus Environment
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absence and presence of c-di-AMP, respectively. The linear relationship (R2 = 0.9926) of the (IF IF0)/IF0 value (denoted as y1) against the c-di-AMP concentration (denoted as x1) ranged from 1 to 20 μM. The relative standard deviations (RSD) of the (IF IF0)/IF0 value at a concentration of 1 μM were 5.95% (n = 3). The calibration equation was determined to be y1 = (0.79 0.07) x1 (0.06 0.10). The theoretical limit of detection (LOD) and limit of quantification (LOQ) are estimated as 3Sa/b and 10Sa/b while Sa is the standard error of the intercept and b is the slope of calibration curve. Thus, this probe enabled the detection of c-di-AMP with LOD and LOQ corresponding to 0.4 and 1.2 μM, which is lower than those measured from coralyne-based assays (Table S1, Supporting Information).10 Moreover, this high-sensitivity probe has great potential for detecting micromolar concentrations of c-di-AMP in bacteria.29 Although the sensitivity of the A20·coralyne probe is inferior to that of the enzyme-linked immunosorbent assay,9 the RNA-based fluorescent sensor,12 and HPLC with mass spectrometry,30-31 it provides numerous advantages, including a short analysis time (within 1 min), low cost, simplicity, and no need for biotin-modified c-di-AMP (Table S1, Supporting Information). The effect of the salt concentration and solution pH on the sensitivity of the A20·coralyne probe was next tested. When the NaCl concentration varied from 0 to 100 mM, c-di-AMP was still capable of removing coralyne from the A20·coralyne probe (Figure S1, Supporting 11 ACS Paragon Plus Environment
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Information). Figure S2 (Supporting Information) shows that the A20·coralyne probe was only efficient to detect c-di-AMP under neutral pH conditions. This is probably due to the fact that the fluorescence intensity of FAM is highly dependent on solution pH. Evidently, this probe is capable of detecting c-di-AMP under high-ionic-strength and neutral pH conditions. Next, this study evaluated the selectivity of the A20·coralyne probe toward c-di-AMP, and Figure 2D shows that change in the (IF IF0)/IF0 value of the A20·coralyne probe occurred within 1 min after separately adding c-di-AMP, c-di-GMP, diadenosine phosphates, and other nucleotides. Notably, c-di-GMP is also identified as a universal bacterial second messenger.32 Only c-di-AMP caused a remarkable increase in the (IF IF0)/IF0 value, indicating that the A20·coralyne probe is highly responsive to c-di-AMP. Previous studies have reported observations of a high level of c-di-AMP (> 10 M) in E. Coli strains expressing disA9 and S. aureus containing one mutation.31 Corrigan et al. demonstrated that the intracellular concentration of c-di-AMP gradually increased from 2 M (OD600nm of approximately 0.9) to 8 M (OD600nm of approximately 10) during the growth of S. aureus.29, 31 Thus, we suggest that the A20·coralyne probe is sensitive enough to detect c-di-AMP in bacteria. To test its practicability, the proposed probe was utilized to determine c-di-AMP in S. aureus (OD600nm of approximately 2.65). Note that many Gram-positive bacteria, such as S. 12 ACS Paragon Plus Environment
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aureus31 and Streptococcus pyogenes,33 are capable of producing c-di-AMP. However, fluorescence from bacterial lysate seriously interfered with the quantification of c-di-AMP by the proposed probe (Figure S3, Supporting Information). This interference is attributed to the intrinsic fluorescence (535 nm) of flavins in the bacteria.34 To circumvent this problem, cyanine 3 and BHQ2 were used in place of FAM and BHQ1, respectively. As indicated in Figure S4 (Supporting Information), the quantification of c-di-AMP by the cyanine 3- and BHQ2-labeled oligonucleotide probe reveals that the linear range was observed between 1 and 10 M. Thus, this probe exhibited similar sensitivity as the FAM- and BHQ1-labeled oligonucleotide probe. When samples of S. aureus were spiked with standard c-di-AMP at different concentrations, a progressive increase in the fluorescence of cyanine 3 at 570 nm occurred, with the increasing concentration of c-di-AMP (Figure 3). A linear calibration curve was obtained by plotting the value of (IF2 IF1)/IF1 (denoted as y2) against the spiked concentration of c-di-AMP (denoted as x2; inset in Figure 3). IF1 and IF2 are the fluorescence intensities of the probe at 570 nm before and after the addition of c-di-AMP, respectively. The measurements of c-di-AMP fitted with a correlation coefficient of 0.9970 to a line with the equation of y2 = (0.14 0.01) x2 + (0.66 0.02). By applying the standard addition method, the concentration of c-di-AMP in S. aureus (OD600nm of approximately 2.6) was determined to be 3.1 0.1 13 ACS Paragon Plus Environment
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M, which is consistent with previously reported results.29 The mean recoveries for c-di-AMP at three spiked levels (1, 5, and 10 μM) were in the range of 93103%. Besides, the concentration of c-di-AMP in bacteria lysate was determined using coralyne as a fluorescent probe in the presence of 250 mM KBr.10 Apparently, coralyne as a fluorescent sensor was incapable of detecting low concentration of c-di-AMP in bacteria lysate (Figure S5, Supporting Information). We suggest that the complexation of coralyne with A20 could be effective to reduce matrix effect from bacteria lysate. These results strongly suggest that the proposed probe is universal and well-suited for the routine analysis of c-di-AMP in bacteria. Sensing of Ap5A. The successful probing of c-di-AMP by the A20·coralyne probe indicates that it can be used for the sensitive and selective detection of other diadenosine nucleotides. As such, Ap5A containing two adenosine moieties was used in place of c-di-AMP. The A20·coralyne probe was then subjected to variable concentrations of Ap5A at fixed 1-min time intervals. The fluorescence of the proposed probe and the (IF4 IF3)/IF3 value incrementally increased with an increase in the concentration of Ap5A (Figure 4A, inset). IF3 and IF4 represent the fluorescence intensities of FAM at 520 nm, as measured from the A20·coralyne probe in the absence and presence of Ap5A, respectively. By plotting the (IF4 IF3)/IF3 value (denoted as y3) versus the Ap5A concentration (denoted as x3), a linear range (R2 = 14 ACS Paragon Plus Environment
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0.9947) for quantifying Ap5A was observed from 10 to 100 μM. The RSD of the (IF4 IF3)/IF3 value at a concentration of 20 μM Ap5A was determined to be 12% (n =3). The equation of calibration curve obtained was y3 = (0.0056 0.0001) x3 + (0.0004 0.0068). Ap5A could be detected with a LOD and LOQ corresponding to 4 and 12 μM, respectively. When the incubation time interval was set to 1 min, this study evaluated the selectivity of the proposed probe toward diadenosine polyphosphates. Interestingly, diadenosine phosphates showed the following trend in the (IF4 IF3)/IF3 value: diadenosine triphosphate > diadenosine tetraphosphate > Ap5A (Figure 2D). This result suggests that the length of the polyphosphate chain strongly affects the ability of diadenosine polyphosphate to remove coralyne from the A20·coralyne probe. This is likely because a long polyphosphate chain facilitates two adenine bases to coordinate with coralyne. A previous study reported the normal concentration of Ap5A in human tears to be 0.036 0.030 μM and an increase in the level (12.45 0.029 μM) of Ap5A occurred in patients with dry eye.35 Thus, we reasoned that the A20·coralyne probe could be utilized to identify patients with dry eye. The dependence of the A20·coralyne probe response to Ap5A was tested in human tear samples. The composition of human tears primarily includes 100 mM NaCl, 26 mM sodium bicarbonate, 16 mM KCl, 5 mM urea, 3 mM NH4Cl, 2.5 mM lactic acid, 31 mM citric acid, 8 μM vitamin C, 3.94 g/L 15 ACS Paragon Plus Environment
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albumin, 2.75 g/L γ-globulin, and 1.7 g/L lysozyme.36-37 Additionally, previous studies have indicated that c-di-GMP and c-di-AMP is only present in bacteria but not in mammals,38-39 suggesting that the proposed probe can be used to detect Ap5A in human tears without the interferences of c-di-GMP and c-di-AMP. To mimic the conditions that such a sensor is used for the determination of Ap5A in dry-eye patients, a series of concentration (10100 M) of Ap5A were spiked into human tears as a mimic system. As indicated in Figure 4B, progressive increases in the fluorescence of the A20·coralyne probe and the (IF4 IF3)/IF3 value were observed after the human tear samples were spiked with a series of concentrations of standard Ap5A. Plotting the (IF4 IF3)/IF3 value (denoted as y4) versus the spiked Ap5A concentration (denoted as x4) yielded a linear calibration curve (R2 = 0.9965) ranging from 10 to 100 μM, with an equation of y4 = (0.008 0.0003) x4 + (0.05 0.019). The mean recoveries for Ap5A at three spiked levels (10, 60, and 100 μM) ranged between 95101%. We determined the LOD of Ap5A in human tears to be 7 μM, which is lower than the level of Ap5A in human tears collected from dry-eye patients. These results suggest that the proposed probe has great potential for discriminating between normal and dry eye groups. The Interaction of Diadenosine Nucleotides with the A20·Coralyne Probe. Previous studies have demonstrated that the binding of coralyne to A20 or c-di-AMP 16 ACS Paragon Plus Environment
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can be investigated by monitoring the absorption and fluorescence spectrum of coralyne.10, 40-42 In an aqueous solution, coralyne aggregates exhibited the maximum absorption peaks at 420 nm and weak fluorescence centered at 475 nm. The separate addition of A20 and c-di-AMP to a solution of coralyne both induced the formation of two new absorption bands and an increase in the fluorescence.10, 40 Thus, we suggest that the analysis of coralyne by absorption and fluorescence spectroscopy is inappropriate to be used to demonstrate diadenosine nucleotide-induced removal of coralyne from the probe. To overcome this problem, the measurement of coralyne by fluorescence polarization and CD was performed before and after the addition of diadenosine nucleotide (c-di-AMP and Ap5A) to the A20·coralyne probe. Figure 5A reveals that coralyne exhibited low fluorescence polarization value (P value) in a solution of 50 mM phosphate (pH 7.5), reflecting that free coralyne rotates at very high speed.43 The incubation of 1 μM A20 to a solution of 5 μM coralyne generated a relatively high P value, indicating that A20 indeed complexes with coralyne and restrict it molecular mobility. As 50 M c-di-AMP was added to the A20·coralyne probe, the obtained P value fell in between those of coralyne and the A20-coralyne complex. The reduction in the P value reflects the conversion of the A20-coralyne complex to the c-di-AMP-coralyne complex because the molecular weight of A20 is much higher than that of c-di-AMP. The CD technique was next used to support the 17 ACS Paragon Plus Environment
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fact of c-di-AMP-induced removal of coralyne from the probe. The analyses of A20, c-di-AMP, and a mixture of c-di-AMP and A20 by the CD technique shows that there was no peak observed in the region of 310 to 350 nm (curve ac in Figure 5B). The A20-coralyne complex generated a positive CD band in the region of 310 to 350 nm (curve d in Figure 5). Obviously, coralyne is effective to induce conformational change of A20. Similarly, Persil et al. demonstrated that the binding of coralyne to polydeoxyadenosine resulted in a new band between 310 and 350 nm.44 However, a solution of the A20·coralyne probe containing 50 μM c-di-AMP exhibited a relatively weak CD band in the same region, signifying that the presence of c-di-AMP reduces the degree of the binding of coralyne to A20 (curve e in Figure 5). These findings strongly suggest that c-di-AMP can remove coralyne from the probe through the coordination of c-di-AMP and coralyne. Moreover, we demonstrated the interaction of Ap5A and the A20·coralyne probe using the same techniques. Compared to c-di-AMP, the addition of Ap5A to the A20·coralyne probe led to relatively small changes in the P value and CD spectra (curve f in Figure 5). This provides strong evidence that the binding strength of the coralyne-c-di-AMP complex is higher than that of the coralyne-Ap5A complex. This result is consistent with the aforementioned description in which the proposed probe provided higher sensitivity and selectivity toward c-di-AMP than Ap5A. 18 ACS Paragon Plus Environment
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To estimate the binding constant between c-di-AMP, the fluorescence of coralyne was monitored as a function of the concentration of c-di-AMP (Figure S6, Supporting Information). Based on results from a Scatchard-plot-fitting McGheevon Hippel analysis,45 the binding constant for coralynec-di-AMP complexation was calculated to be 1.0 107 M−1 (Figure S6, Supporting Information). In contrast, a previous study showed that the binding constant between coralyne and A20 was 4.0 104 M−1.40 Apparently, the interaction between c-di-AMP and coralyne is stronger than the A20 binding to it, thus facilitating the c-di-AMP-induced removal of coralyne from the A20·coralyne probe.
CONCLUSION This study demonstrated that the A20·coralyne probe based on the competitive binding of A20 and diadenosine nucleotide to coralyne is characterized by ease of operation, high sensitivity, and excellent specificity for recognizing c-di-AMP in bacteria lysate and Ap5A in human tears. In addition, this sensing technique can be performed at room temperature and completed within 1 min. To the best of our knowledge, this is first example of the use of an oligonucleotide-based fluorescence probe for the sensitive and selective detection of c-di-AMP and Ap5A. Compared to a coralyne-based sensor that is unable to detect c-di-AMP in bacteria lysate, the 19 ACS Paragon Plus Environment
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proposed probe has been used for the determination of c-di-AMP level in the same real samples. Although the sensitivity of the proposed probe toward c-di-AMP is not comparable to that of RNA-based fluorescent sensors,12 we suggest that the sensitivity of this probe should be further improved by determining the optimal fluorophore-quencher combination. These findings strongly suggest that the proposed probe could be applied for determining c-di-AMP in any type of bacteria. The synthesis of c-di-AMP by diadenylate cyclase DisA further suggests that the proposed probe could be suitable for detecting the activity and inhibition of diadenylate cyclase DisA.10-11
ACKNOWLEDGEMENTS We would like to thank the Ministry of Science and Technology (NSC 100-2628-M-110-001- MY4) for the financial support of this study.
ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following files are available free of charge on the ACS Publications website at DOI:
Additional Figures, including the effects of solution pH and salt concentration 20 ACS Paragon Plus Environment
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on the sensitivity of the probe toward c-di-AMP, the quantification of c-di-AMP by cyanine 3- and BHQ2-labeled oligonucleotide probe, the determination of c-di-AMP in bacteria lysate by coralyne, and the determination of binding constant between diadenosine nucleotide and coralyne.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] Phone:
011-886-7-5254644
Fax:
011-886-7-3684046. Note The authors declare no competing financial interest
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Figure 1. Turn-on fluorescence sensing of c-di-AMP in bacterial cell lysates and Ap5A in human tears based on the removal of coralyne from the A20·coralyne probe.
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Figure 2. Sensing of c-di-AMP. (A) Fluorescence intensity of the A20·coralyne probe at 520 nm as a function of the concentration of coralyne. (B) Time-dependent fluorescence changes at 520 nm of the A20·coralyne probe in the presence of 5 μM c-di-AMP. (C) Fluorescence spectra changes observed upon the incubation of the A20·coralyne probe with different concentrations of c-di-AMP for a fixed time interval of 1 min. Inset: a plot of the (IF IF0)/IF0 value versus the concentration of c-di-AMP. (D) The (IF IF0)/IF0 value obtained from the addition of (a) c-di-AMP, (b) cyclic diguanylate monophosphate, (c) adenosine monophosphate, (d) adenosine diphosphate, (e) adenosine triphosphate, (f) guanosine monophosphate, (g) guanosine triphosphate, (h) cytidine triphosphate, (i) thymidine triphosphate, (j) diadenosine triphosphate, (k) diadenosine tetraphosphate, and (l) Ap5A. The concentrations of c-di-AMP and cyclic diguanylate monophosphate are 10 μM, while the concentrations of other analytes are 100 μM. The incubation time is 1 min. (AD) The error bars represent standard deviations based on three independent measurements.
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Figure 3. Determination of c-di-AMP in S. aureus by the A20·coralyne probe. Samples of bacterial lysate were spiked with different concentrations of c-di-AMP. The arrow indicates the signal changes as increases in the c-di-AMP concentration (0, 1, 2, 4, 5, and 10 μM). Inset: a plot of the (IF2 IF1)/IF1 value versus the concentration of c-di-AMP. The incubation time is 1 min. The error bars represent standard deviations based on three independent measurements.
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Figure 4. (A) Fluorescence spectra changes observed upon the incubation of the A20·coralyne probe with different concentrations of Ap5A for 1 min. Inset: a plot of the (IF4 IF3)/IF3 value versus the concentration of Ap5A. (B) Turn-on fluorescence sensing of the spiked Ap5A in human tear by the A20·coralyne probe. Samples of human tear were spiked with different concentrations of Ap5A. Inset: a plot of the (IF4 IF3)/IF3 value versus the concentration of Ap5A. The incubation time is 1 min. (A, B) The error bars represent standard deviations based on three independent measurements. 32 ACS Paragon Plus Environment
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Figure 5. Characterization of the binding of coralyne to c-di-AMP and Ap5A in the presence of A20. (A) Fluorescence polarization values of (a) coralyne, (b) coralyne and A20, (c) the A20·coralyne probe and c-di-AMP, and (d) the A20·coralyne probe and Ap5A. The concentrations of coralyne, A20, c-di-AMP, and Ap5A are 5, 1, 50, and 100 μM, respectively. (B) CD spectra of (a) A20, (b) c-di-AMP, (c) c-di-AMP and A20. (d) coralyne and A20, (e) the A20·coralyne probe and c-di-AMP, and (f) the A20·coralyne probe and Ap5A. (B) The concentrations of coralyne, A20, c-di-AMP, and Ap5A are 50, 10, 50, and 100 μM, respectively. (A, B) The reaction was performed in 50 mM phosphate (pH 7.5) for 30 min at ambient temperature. 33 ACS Paragon Plus Environment
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