Article pubs.acs.org/ac
Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization Anna Shahmuradyan and Ulrich J. Krull* Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada S Supporting Information *
ABSTRACT: Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes. fluorescence emission of the dye molecules. Upon hybridization of the probe with complementary target, the dimer dissociated interrupting the excitionic interactions and resulting in enhancement of fluorescence emission due to intercalation of the dye molecules into the hybrid structure.9,10 This approach to transduction of hybridization makes use of intrinsically labeled oligonucleotide probes that allow reversible determination of unlabeled target in a single step and eliminates need for additional reagents such as those found in sandwich assays or in staining of hybrids using intercalators. The work reported herein explores the analytical performance of the general configuration shown in Figure 1, where the concept of use of intrinsically labeled probes is combined with optical excitation by quantum dots (QDs). The DNA probes are immobilized on the surface of QDs, and the fluorescent dyes are excited via fluorescent resonance energy transfer (FRET) from the QD donors. This configuration offers several
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yanine dyes have been widely used for a variety of biological and biomedical applications involving nucleic acids, such as staining of DNA in agarose gel and capillary electrophoresis separations,1,2 detection of nucleic acids after high-performance liquid chromatography separations,3 labels in polymerase chain reaction,4 and acceptors in fluorescence resonance energy transfer (FRET).5,6 The properties that make cyanine dyes useful in such applications include high affinity for double-stranded nucleic acids, large extinction coefficients associated with strong π−π absorption, and quantum yield that increases by orders of magnitude upon intercalation into double-stranded DNA.7 Cyanine dyes can form aggregates in aqueous solutions due to π stacking interactions that are associated with the polarizability and hydrophobicity of the dyes.8 In such aggregates, the fluorescence is quenched due to excitonic interactions of the transition dipoles. This optical property of the aggregates offers potential for development of DNA detection bioassays based on an “on−off” switching system as reported by Okamoto and co-workers.9,10 Singlestranded oligonucleotide probes were labeled with two adjacent derivatives of thiazole orange dye that formed an H-aggregate dimer in aqueous solution, resulting in quenching of the © XXXX American Chemical Society
Received: November 30, 2015 Accepted: February 11, 2016
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EXPERIMENTAL SECTION A detailed description of the experimental procedures, instrumentation, and data analysis can be found in the Supporting Information. Reagents and Oligonucleotides. Qdot 525 ITK Streptavidin Conjugate Kits were from Life Technologies, a ThermoFisher brand. Oligonucleotides were from Integrated DNA Technologies (Coralville, IA) and were purified by either standard desalting or HPLC by the manufacturer. The oligonucleotide sequences were dissolved in autoclaved MilliQ water (purified water from a Milli-Q cartridge filtration system with a resistivity of 18.2 MΩ·cm) and stored at −20 °C. 2-Methylbenzoxazole (C8H7NO, 99%), 5-bromovaleric acid (Br(CH2)4COOH, 97%), 4-dimethylamino benzaldehyde ((CH3)2NC6H4CHO, ≥99.0%, HPLC), acetic anhydride ((CH3CO)2O, ReagentPlus, ≥99%) N-hydroxysuccinimide (NHS, C4H5NO3, 98%), 1,2-dichlorobenzene (C6H4Cl2, anhydrous, 99%), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, C 8 H 17 N 3 ·HCl, purum ≥98%), and goat serum were from Sigma-Aldrich (Oakville, ON). All buffer solutions were prepared using a deionized water purification system (Milli-Q, 18 MΩ·cm) and were autoclaved prior to use. The buffer solutions included 100 mM tris-borate buffer (TB, pH 7.4) and 100 mM bicarbonate buffer (BIC, pH 8.3). Synthesis of OD539 (3-(4-Carboxybutyl)-2-[4(N,Ndimethylamino)styryl]benzoxazolium). The synthesis of this dye has been reported elsewhere.10 Briefly, 2-methylbenzoxazole (3.00 mL, 25.0 mmol) and 5-bromovaleric acid (9.00 mg, 50 mmol) were suspended in 10 mL of 1,2dichlorobenzene. The resulting mixture was stirred and heated at 120 °C overnight. The reaction mixture was cooled to room temperature, and 200 mL of dichloromethane was added. The resulting suspension was stirred at room temperature for 2 h. The precipitate was then filtered, washed, and dried resulting in white powder. The white powder (312 mg, 1.00 mmol) was then mixed with 4-dimethylaminobenzaldehyde (150 mg, 1.00 mmol) and suspended in 10 mL of acetic anhydride. The suspension was heated and stirred at 120 °C for 30 min. At the end of the 30 min, 10 mL of purified water (Milli-Q, 18.2 MΩ· cm) was added and the resulting solution was heated for another 30 min. The solvent was then evaporated at reduced pressure, and 100 mL of acetone was added to the residue. The resulting suspension was allowed to stand at room temperature for 30 min. The precipitate was filtered, washed with acetone, and dried under reduced pressure giving a reddish brown powder. The mass of the synthesized dye was found to be 365.06 ([M − Br]+) using ESI MS. The resulting dye (8.9 mg, 20 μmol) was mixed with N-hydroxysuccinimide (4.6 mg, 40 μmol) and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (7.7 mg, 40 μmol) in 0.5 mL of dimethylformamide and stirred at room temperature overnight. The resulting clear solution was stored in a freezer. Attaching Dye to the DNA Probe. DNA probe was from Integrated DNA Technologies (IDT) and was provided with two internal amine linkers, AmC6dT or UniLink, and a biotin attached to the 5′ end. AmC6dT is a modified nucleotide with an amine attached to the nucleobase by a 6 carbon linker. UniLink has an amine that is attached to the phosphate backbone by a 6 carbon linker. The dye solution was added to the diamino modified DNA probe at 50 mol equivalents in 100 mM bicarbonate buffer at pH 8.3. The resulting solution was
Figure 1. Schematic representation of the dye labeled probe on the surface of a QD. The dye is excited by FRET with the QD serving as donor. (a) In the absence of the target, the dye molecules form an Haggregate, which results in suppression of fluorescence emission due to excitonic interaction. (b) The hybridization of the target results in the disruption of the excitonic interaction and strong emission from intercalated dye.
advantages to the analytical performance. The QDs have broad absorption and narrow emission bands, so that a range of absorption wavelengths can be easily converted into a welldefined emission for the FRET process. FRET-based detection provides inherent spatial selectivity as FRET efficiency is distance dependent. The FRET excitation interrogates a distance on the scale of 10 nm (eq 1), so that only the surface interactions on the QDs are sampled. E=
aR o6 r 6 + aR o6
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(1)
where E is the FRET efficiency, r is the distance between the donor and the acceptor, a is the total number of the acceptors, and Ro is the Förster distance, the distance at which the energy transfer has 50% efficiency.11 Other examples of QD-nucleotide sensors have been previously reported where QDs were used as FRET donors in paper-based solid-phase nucleic acid hybridization assays,12 for detection of RNA13 and microRNA14 as well as for construct beyond sensors.15,16 The interdependence of QD and dye emission allows for ratiometric detection and opportunities for improved efficiency of hybridization based on the high curvature of the nanoparticle surface, multiplexed analysis using concurrent spectroscopic color channels, and signal amplification by proximity of an acceptor to multiple donors in solid-state assay formats. Ratiometric detection is based on the calculation of the emission intensity ratio of the donor and the acceptor and offers improved precision while accounting for donor photoluminescence (PL) quenching and sensitization of acceptor PL.12 This investigation of the FRET-based transduction system considers a variety of structural motifs associated with the location of the cyanine dyes to extend the original work of Okamoto and co-workers.9,10 Permutations include investigation of placement of the dyes on nucleobases and on the phosphate backbone and the effect of the distance of separation of dyes. B
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Analytical Chemistry shaken using a vortex mixer and left overnight (at 4 °C). The solution was then centrifuged at 8000 rpm for 3 min. The supernatant was collected and purified using a NAP5 column. The concentration of the DNA probe was determined using the DNA absorption band at 260 nm (Molar Extinction Coefficients were provided by IDT). Preparation of QD−Probe Conjugates. The doubly labeled DNA probes were mixed with streptavidin coated green QDs at 70 equiv in 100 mM Tris borate (TB) buffer with 20 mM NaCl at pH 7.4. These QDs accommodate on average 46 probes per QD.17 The solution was mixed on a shaker platform for 45 min. 100 kDa spin filters were used to remove excess DNA probes in solution. The immobilization of the DNA probes was confirmed using gel electrophoresis (Supporting Information). Positioning of Dyes on Oligonucleotide Probes. Two types of probes were ordered from IDT; one contained two thymine bases each with amino modified linkers (Amino Modifier C6 dT) and the other was modified with two UniLink Amino Modifiers. The modified thymine bases were adjacent, located either in the middle of the probe sequence or one-quarter of the distance from the terminus of the oligonucleotide sequence. The points for dye attachment were either immediately next to each other or separated by one nucleotide. The dye-labeled probes were conjugated to the QDs, and the resultant QD−probe conjugates were incubated with fully complementary (FC) target sequences at 2 μM concentration for 2 h in a TB buffer with 100 mM NaCl. A summary of the probe sequences is shown in Table 1.
between 500 and 600 nm. The selectivity of the assay was evaluated by comparing the PL for fully complementary (FC) target with one containing a base pair mismatch (1BPM). The target with 1BPM was incubated with the QD−probe conjugate in a 100 mM TB buffer at pH 7.4 with 100 mM NaCl for 2 h. The effect of the location of the 1BPM was investigated by placing the dye and mismatch at various distances with up to 3 nucleotides of separation. This experiment was repeated in the presence of 75% v/v goat serum.
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RESULTS AND DISCUSSION The absorption and emission spectra of the doubly labeled probes were measured in the presence and the absence of the fully complementary target. Two absorption bands were present in both cases in a range of 400−550 nm (Figure 2a). The band centered with the shorter wavelength (482 nm) was more prominent when the probes were in the single-stranded state. The longer wavelength absorption band (520 nm) dominated when the fully complementary target hybridized to the probe. This latter absorption band was also prominent in the spectrum of a probe labeled with only a single dye molecule (Supporting Information). The difference in the photophysical properties of the dyes in the presence and the absence of the target are due to the formation of a dimeric structure between the dyes.9 The self-association of the dye molecules in aqueous solutions occurs due to π stacking interactions between the conjugated systems of the dyes, which minimizes exposure to water.8 The shift toward a shorter wavelength upon formation of a dimeric structure is a characteristic of H-aggregates. The formation of the H-aggregate allows for excitonic interaction between the dye molecules, which in turn suppresses fluorescent emission10 (Figure 2b). Exciton coupling theory suggests that the dye molecules are treated as point dipoles and the excited state splits into two energy levels due to the interaction of transition dipoles.18 The higher energy level results from the in-phase alignment of the two dipoles, whereas the out-of-phase alignment gives rise to the lower energy level.18 Absorption of an incoming photon results in the transition to the upper excitonic state followed by rapid deactivation to the nonemissive lower excitonic state.18 Hybridization of the fully complementary target results in the dissociation of the dimer, leading to the disruption of the excitonic interaction and restoration of the fluorescence emission. Fluorescence intensity is directly affected by the quantum yield, and the quantum yield is expressed by the ratio of the sum of the radiative relaxation over the sum of the nonradiative relaxation rate constants.11 The disruption of the excitonic interaction leads to an increase in the quantum yield and the subsequent increase in fluorescence intensity. The quantum yield of the probe−target hybrid was 1.6 times the quantum yield of the probe in the single-stranded state which is lower than expected for a derivative of a thiazole orange dye in a similar environment.9 The relatively small change in the quantum yield may be a consequence of incomplete dissociation of the dimer upon hybridization of the target. The presence of two emissive populations, dimer and monomer, was evident in lifetime measurements (Supporting Information). The fluorescence decay profile of the probe in the double stranded state was fit well with a double exponential function, whereas the probe in the single stranded state could be fit to a single exponential function. The presence of the dimeric form is also supported by the absorption spectra. The shorter wavelength absorption band is present as a shoulder in
Table 1. SMN1 Oligonucleotide Sequences Used in the Assaya name 50-1 Probe 50-1 FC tgt 50-1 1BPM-0 tgt 50-1 1BPM-1 tgt 50-1 1BPM-2 tgt 50-1 1BPM-3 tgt 25-1 Probe 25-0 Probe 25-1 FC tgt
sequence 5′ Biotin-TTG ATT T/Amine/G/Amine/CT GAA ACC C 3′ 5′ TAC TGG CTA TTA TAT GGG TTT CAG ACA AAA TCA A 3′ 5′ TAC TGG CTA TTA TAT GGG TTT CAC ACA AAA TCA A 3′ 5′ TAC TGG CTA TTA TAT GGG TTT CTG ACA AAA TCA A 3′ 5′ TAC TGG CTA TTA TAT GGG TTT AAG ACA AAA TCA A 3′ 5′ TAC TGG CTA TTA TAT GGG TTT CAG ACA AAA GCA A 3′ 5′ Biotin-ATT T/Amine/G/Amine/CT GAA ACC CTG T 3′ 5′ Biotin-ATT/Amine//Amine/GCT GAA ACC CTG T 3′ 5′ TCC TTT ATT TTC CTT ACA GGG TTT CAG ACA AAA T 3′
a
tgt = target, FC = fully complementary, 1 BPM = 1 base pair mismatch, NC = noncomplementary, and /Amine/ = modified thymine (AmC6dT) or UniLink. The mismatched base in 1 BPM tgt is bolded, italicized, and underlined.
Sensitivity and Selectivity Assays. The 34 and 90 base length target oligonucleotides were from Integrated DNA Technologies (Table 1). Calibration curves were constructed using solutions of 0.01 to 2 μM target concentrations. The targets were incubated with the QD−probe conjugates in 100 mM TB buffer with 100 mM NaCl at pH 7.4 for 1−2 h. The quantum dots were excited at 405 nm using a fluorescence spectrometer, and photoluminescence (PL) was measured C
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Figure 2. (a) Normalized absorption and PL of the dye labeled probe measured in the absence and the presence of the FC target. The absorption (dashed line) and PL (solid line) are indicated by red and blue for the nonhybridized probe and the hybrid, respectively. (b) The excited state of the dimer splits into two energy levels due to the interaction of the transition dipoles. The out-of-phase alignment of the dipoles results in the lower energy level, whereas in-phase alignment of the dipoles results in the higher energy level. The transition from the ground state to the lower energy level is forbidden. After excitation to the allowed upper level, rapid internal conversion takes place to the lower level, which is followed by nonradiative relaxation to the ground state.18
Figure 3. Two types of linkages were used to attach the dye to the DNA probe, amine modifier UniLink (a) and an amino modified thymine base (b). The UniLink conjugates the dye to the phosphate backbone of the oligonucleotide, whereas the modified thyme conjugates the dye to the nucleobase. The MFR for the probe containing the modified thymine bases was 5.2 times the MFR of the probe labeled with the UniLink. (c, d) Absorption spectra of the doubly labeled probe in the presence and the absence of the target, as well as of a singly labeled probe for “50-uni” and “501”, respectively.
the spectrum of the double stranded state and absent in the spectrum of the probe labeled with a single dye (Supporting Information). The PL associated with the hybridization of the target to the probe was quantified using a ratiometric approach based on a modified FRET ratio (MFR) (Supporting Information). The
change in the magnitude of the MFR provided an indication of the effect of the type of linkage used to attach the dye to the probe and the location of the dye within the probe sequence, as well as the distance between the dyes in the presence and the absence of the target. D
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Figure 4. Signal from the dye in the presence of the FC target was not significantly impacted due to variation of the distance between the locus of the dyes and the QD. In one configuration, the dye was attached to the center of the probe (50-AmC6dT, a), and in another configuration, it was attached a quarter of the distance along the probe length (25-AmC6dT, b). The dyes were attached using the amine modified thymine, C6dT. The ratio between the MFR of the 50-AmC6dT probe and the 25-AmC6dT probe was 1:1.
Figure 5. (a, b) The effect of the distance between two dyes was investigated by attaching the dyes to nucleotides that were immediately adjacent (25-0) or that were separated by one nucleotide (25-1). The dyes were attached to the probe one-quarter of the way along the length of the sequence using the amine modified thymine AmC6dT. (c, d) Absorption spectra for the probe in the single stranded state, double stranded state, and a single dye molecule for “25-1” and “25-0”.
The type of linkage used to attach the dye to the probe had a significant effect on the PL of the dye. There were two types of commercially available linkages used; both contained a six carbon aliphatic spacer arm. A Uni-Link amino-modifier provided for attachment of an amine to the phosphate
backbone of an oligonucleotide sequence (Figure 3). A modified thymine nucleobase, AmC6dT, provided for conjugation to the nitrogenous base. The MFR of the probe containing the AmC6dT (50-1 probe) was 4.6 times higher than the MFR of the probe containing the Uni-Link(50-uni E
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Figure 6. (a) Concentration−response showing MFR response to 34mer and 90mer FC target. (b) Fluorescence emission spectrum corresponding to a, QD; b, QD−probe conjugate; c−k increasing concentrations of target.
only as a shoulder after the addition of the fully complementary target, which indicates that the excitonic interaction was not sufficiently disrupted (Figure 5c,d). The quantitative response of the assay was investigated using FC targets of 34mer and a 90mer length. In case of the 34mer target, the response increased linearly with target concentration ranging from 0.01 μM (2 pmol) to 0.75 μM (150 pmol), corresponding to a dynamic range of 1 order of magnitude (Figure 6). The limit of detection (LOD) of the assay was determined experimentally to be 2 pmol. In the case of the 90mer target, the dynamic range, the detection limit, and the magnitude of the signal were similar to that of the 34mer target (Figure 6). The similarity of hybridization efficiency of the shorter and longer targets is attributed to the angle of deflection between the strands provided by the radius of curvature of the nanoparticle interface.20 The increase in distance between the strands on a surface of high curvature reduces steric interactions, thus facilitating hybridization efficiency.20 The quantitative response of the assay was also investigated in a complex matrix consisting of 75% v/v goat serum (Supporting Information). Despite some reduction of signal intensity, the dynamic range and the limit of detection were found to be similar to those obtained in buffer solution. The primary intention of this work was to integrate intercalating dyes into single-stranded oligonucleotide probes in a manner that could be used for transduction of hybridization by a QD-FRET mechanism. A further opportunity involving improvement of detection level was examined. Excitonic interaction between the dye molecules in the doubly labeled probes caused quenching of the fluorescence, thus reducing background. The resultant increase in the difference between the signals obtained from the hybridized and the nonhybridized states allowed for improvement of the limit of detection. There was higher background in the case of the singly labeled probes, and the LOD was improved by 5 with doubly labeled probes (Supporting Information). In addition, the sensitivity (slope of response) using the doubly labeled probes was higher than that observed for the singly labeled probes (Supporting Information). The sensitivity of the configuration containing QDs and doubly labeled probes was also compared to the sensitivity of a system in the absence of the QDs where the doubly labeled probes were excited directly by the excitation source (Supporting Information). Results showed a 34% increase in the sensitivity of the system for probes that were excited using FRET from QD donors, with equivalent LOD, indicating that the advantages of QD-FRET excitation were achieved without sacrificing analytical perform-
probe) (Figure 3a). The lower PL of the UniLink probes could result from steric restrictions imposed on the dyes. Unlike the dyes directly attached to the nucleobases, the dyes attached to the DNA via the UniLink spacer might not have sufficient range of motion, hence preventing the dyes from intercalating into the DNA duplex. This is evident in the absorption spectrum of the “50-uni” probes as the characteristic long wavelength absorption peak of the monomeric state emerged as only a shoulder after the hybridization of the fully complementary target, and the short wavelength absorption band that is characteristic of the dimeric state remained prominent (Figure 3c,d). The location of the dye within the probe sequence was chosen relative to the 5′ end of the sequence (Figure 4) so that FRET efficiency would be maintained. In the case of the probes referred to as “25-1”, the dyes were attached to the fifth and the seventh nucleotides, and this represents one-quarter of the length of the sequence. The probes referred to as the “50-1” located the conjugated dyes in the middle of the sequence. On the basis of eq 1, the location of the dye with respect to the QD was expected to have a significant effect on the signal intensity. However, there were no significant differences between the MFR for the two dye configurations. In order to rule out the possibility of the probes collapsing on the surface of the QDs, unlabeled probes of the same sequence were hybridized with targets labeled with Cy3 dye at the 3′ and the 5′ end. The FRET ratio observed for the proximal target was 6 times the FRET ratio of the distal target, which suggests that the probes do not fold onto the nanoparticle surface. The results shown in Figure 4 suggest that any increase in the FRET efficiency due to a closer proximity of the dye to the donor was largely offset by weakened intercalation of the dyes due to being close to the terminus of the hybrid sequence.19 The signal intensity of the dye is expected to increase upon hybridization of the target as it causes disassociation of the nonemissive dimer; however, it was previously noted that partial intercalation of the dye caused only a 50% increase in the signal intensity.19 The distance between the dyes had a significant effect on the signal intensity. The MFR of the probes labeled with dyes that were separated by one nucleotide (25-1 probes) was a factor of 4.5 greater than the MFR of the probes labeled with dyes that were immediately adjacent (25-0 probes) (Figure 5a,b). It was anticipated that increasing the distance between the dyes would weaken the excitonic interaction between the dyes, and this was supported by the absorption spectra. In the case of the “25-0” probe, the short wavelength absorption band did not reduce in intensity and the longer wavelength absorption band emerged F
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Figure 7. Selectivity of the assay was evaluated by comparing the MFR of FC and a 1BPM target. (a) The mismatch was located next to the labeled nucleotide (Table 1). (b) The absorption spectrum of the 1BPM does not have the characteristic peak of a probe labeled with a single dye molecule at 520 nm. (c) With increasing distance between the mismatch and the dye, the signal intensity and the MFR increased.
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CONCLUSIONS An intrinsically responsive FRET transduction method to detect hybridization was investigated using dye labeled oligonucleotide probes that were immobilized on the surface of QDs. Two commercially available linkers were assessed for concurrent conjugation of two identical thiazole orange derivatives at various locations on single-stranded probe oligonucleotide. Amino modified thymine provided for better signal-to-noise in comparison to attachment of dyes to the phosphate backbone of oligonucleotide probes. It was shown that attachment of the dyes closer to the QDs did not result in the improvement of the signal for the relatively short oligonucleotide probes that were used. This was attributed to the possibility that the increase in the FRET efficiency due to a decrease in the distance between the donor and acceptor was counteracted by the decrease in fluorescence intensity due to the partial intercalation of the dyes as they were located near the end of the sequence where the hybrid was labile. The impact of the distance between the dyes on signal generation was explored. The dyes were attached either next to each other or separated by one nucleotide. The latter provided for improved signal intensity, as increasing the distance between the dyes further weakened the excitonic interaction so that they could more effectively intercalate into the DNA duplex. The system was also assessed for quantitative response to increasing concentrations of a fully complementary 34mer target and a 90mer target, indicating pmol detection levels with similar response to both sequences even though they differed in length. The selectivity of the assay was investigated using a 1BPM target, establishing that the location of the mismatch was
ance. In addition, the precision in the measurements was improved in the case of direct excitation of the doubly labeled probes. Single nucleotide polymorphism (SNP) discrimination was used as a proxy to evaluate the selectivity of the assay. A sequence was chosen for SNP discrimination in which the 1BPM was located next to the amine modified thymine that was linked to the dye (1BPM-0). The MFR for this probe was −0.08 ± 0.03, indicating that the analytical signal was lower than the background signal that was observed in the absence of the target (Figure 7a). The magnitude of the MFR was observed to behave as expected as the distance between the 1BPM was moved by 1 or 2 nucleotides (Figure 7c) away from the amine modified thymine conjugation sites. The formation of the mismatched base pair in the 1BPM-0 probe results in the decrease in the fluorescence intensity because the mismatched nucleotide located next to the labeled nucleotide serves as a binding site for the dye.9 The mismatched base pair causes the dissociation of the dyes from the DNA structure by lowering the binding affinity to the DNA. The dissociated dyes form a nonemissive bichromophoric aggregate which in turn results in the decrease of fluorescence intensity,9 as is evident from the absorption spectrum of the dye in the presence of the 1BPM target where the characteristic monomeric absorption peak at 520 nm is missing (Figure 7b). The experiment was repeated in the presence of goat serum. The results showed a reduction in signal due to an increase in the background; however, the same trend was observed (Figure S3). G
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(20) Noor, O. M.; Hrovat, D.; Moazami-Goudarzi, M.; Espie, G. S.; et al. Anal. Chim. Acta 2015, 885, 156−165.
important because the mismatch could serve as an interaction site for adjacent dyes.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04536. Description of instrumentation used, characterization of the FRET pair and equations used in the data analysis, gel electrophoresis results, additional absorption spectrum, and SNP discrimination in 75% v/v goat serum solution. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this research. The authors would also like to thank Samantha Tabone for technical assistance with some of the research work and for helpful discussion.
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REFERENCES
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