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Photocontrolled DNA Hybridization Stringency with Fluorescence Detection in Heterogeneous Assays Yunqi Yan, Soumyadyuti Samai, Kristi L. Bischoff, Jie Zhang, and David S Ginger ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.5b00233 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016
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Photocontrolled DNA Hybridization Stringency with Fluorescence Detection in Heterogeneous Assays Yunqi Yan, Soumyadyuti Samai, Kristi L. Bischoff†, Jie Zhang, David S. Ginger* Department of Chemistry, University of Washington, Seattle, 98195 † Mel and Enid Zuckerman College of Public Heath, University of Arizona, Tucson, 85724 * Corresponding Author:
[email protected] Abstract We study hybridization and light-induced dehybridization of azobenzene-modified DNA bound to glass substrates with fluorescently-labeled oligonucleotide targets in solution. We show that fluorescent readout using a commercial array scanner is compatible with azobenzene-modified DNA capture sequences and, importantly, that the fluorescent signals generated using azobenzene-modified sequences are similar to those from azobenzene-free capture strands. In addition, we demonstrate that we can photoswitch azobenzene molecules on a surface in the presence of fluorophores and thus that we are able to control the dehybridization behavior of the immobilized azobenzene-modified DNA with its target sequence in solution. We further study the dehybridization of perfectly-matched target sequences and the single-base-mismatched sequences as a function of radiant fluence. Though both target sequences dehybridize upon exposure to ultraviolet light, we measure higher fluorescent signals after UV irradiation for perfectly complementary sequences compared to those with single-base mismatches, consistent with selective photoinduced dehybridization of partially-mismatched sequences. The demonstration of photoinduced differential dehybridization phenomenon on chip surfaces in the presence of fluorophores indicates that photonic DNA hybridization stringency is compatible with optical readouts in heterogeneous assays.
Keywords: Azobenzene-modified DNA, DNA arrays, DNA assays, Chip, Fluorescence, Photoinduced isomerization
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Control over the hybridization and dehybridization of DNA is essential for processes ranging from PCR1 and gene regulation2,3 to molecular sensing4,5 and bio-inspired materials assembly.6,7 In the laboratory, the hybridization state of DNA is usually manipulated by temperature,8,9 ionic strength,10 or the use of synthetic nucleic acids.11,12 Recently however, the covalent incorporation of molecular photoswitches into oligonucleotides has opened up a route to control DNA hybridization using specific wavelengths of light as an external stimulus.13-16 By covalently linking azobenzene moieties onto the DNA backbone via the d-threoninol bond (Scheme 1a),13,17 Asanuma et al. have shown that light can be used to manipulate the hybridization of azobenzene-modified DNA in a reversible manner18 due to the photoinduced isomerization of the azobenzene moieties from trans to cis form.19,20 As verified by NMR and UV-Vis,14,17 exposure of the azobenzene-modified DNA to UV light (~365 nm, absorbed only by the azobenzene) results in a trans-tocis photoisomerization, which destabilizes the double-stranded DNA.18 Exposing the azobenzenemodified DNA to blue light (~470 nm) reverses the process, allowing the double-stranded DNA to reform.13 Photo-control of hybridization using azobenzene-modified DNA has been demonstrated in applications such as optical regulation of gene transcription21 and polymerase reactions,22 and the creation of photo-reactive biomaterials,23,24 optically-triggered drug delivery25 and enzyme inhibition.26 Recently using azobenzene-modified DNA-functionalized gold nanoparticles, our group showed that photon dose can be used as a variable to control hybridization stringency.24 This effect arises from the variation of the azobenzene quantum yield with the local sequence in a way that local mismatches result in an increase in azobenzene photoisomerization quantum yield,27 and hence a faster decrease in DNA stability upon UV photon irradiation for partially complementary sequences as compared to fully-matched sequences. So far, these experiments have been dominated by homogeneous solution environments, and with limited study of the interaction between fluorescent labels and the azobenzene photoswitches. Here, motivated by the popularity of heterogeneous assays28-30 and fluorescence detection in molecular diagnostics,31,32 we explore the possibility of using azobenzene-modified DNA attached to solid supports,
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in the presence of fluorescently labeled DNA. To this end, we study photoinduced hybridization and dehybridization of azobenzene-modified DNA substrates with fluorophore-attached target sequences. Scheme 1 depicts our approach. Through maleimide-thiol chemistry, 20-mer capture sequences with four internal azobenzene modifications are covalently functionalized onto glass slides which are prior-treated with heterobifunctional molecules and thiol-reactive moieties (see Supporting Information for full sequences). We detect fluorescence from immobilized azobenzene-modified DNA spots after soaking them with the hybridization solution containing rhodamine-labeled target sequences, indicating that the azobenzene-modified sequence is compatible with commercial fluorescent readout. We further study the photocontrolled dehybridization of azobenzene-modified capture sequences and fluorescent targets with slightly-different sequences under continuous 365-nm irradiation. We find that target sequences with a single-base mismatch lead to more rapid dehybridization compared to targets with matched sequences, extending the photonic hybridization stringency to azobenzene-modified DNA chips under heterogeneous environments.
Experimental Section Preparation of DNA slides We adapted the method from O’Ferral et al. to covalently attach thiolated oligonucleotides to glass slides.33 Briefly, plain microscope glass slides (Fisher Scientific) were thoroughly cleaned with 15minutes of sonication in isopropyl alcohol immediately followed by 5-minutes exposure to air plasma. The slides were then modified with an amino-terminated monolayer by soaking in 1% (v/v) 3aminopropyltriethoxysilane ethanolic solution for 20 minutes at room temperature, and were subsequently annealed at ~95-100 °C for 5 minutes. Immediately after heating, the heterobifunctional succinimidyl 4[malemido-phenyl]butyrate (SMPB) solution (10 mM in 1:4 DMSO:ethanol) was kept on top of glass slides for 2 hours at room temperature followed by rinsing with ethanol and drying with nitrogen. Newlycleaved 0.5 µL10 µM thiolated azobenzene-modified DNA was manually spotted on the glass slides using a digital pipette. The spots were left to dry by keeping the DNA solution at ~40% humidity
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(ambient) for 2 hours at room temperature. After attachment, slides were soaked in saline sodium phosphate citrate (SSPC) buffer for 5 minutes before being stored at room condition. The home-made azobenzene-modified DNA slides were used within one week. We also explored multiple attachment chemistries. Amine-terminated oligonucleotides were covalently linked to glass slides via the amine-epoxy reaction by using commercial epoxy substrates. We described the experimental details in the supporting information. Preparation of DNA solutions All oligonucleotides were synthesized and purified by Integrated DNA Technology (IDT) Incorporation. DNA aliquots were prepared right after their arrival and were stored dry in the freezer (-15 °C). The di-thiol protected oligonucleotides were deprotected (10 µM oligonucleotides) with 10 mM DTT in phosphate buffer for 15 minutes before a de-salting step with micro-chromatography columns (Micro Bio-Spin® 6 Columns, Bio-Rad) to yield the free thiol-terminated DNA. Cleaved thiolated oligonucleotides were dissolved immediately in phosphate buffer to be spotted onto glass slides or stored dry temporarily for use within 3 days. Fluorophore-labeled oligonucleotides were dissolved in desired amount in 10 mM phosphate buffer with 0.01M NaCl and 0.02% azide and were stored in buffer in a refrigerator (~4 °C) for less than one month before use. Hybridization experiment Glass slides bearing azobenzene-modified capture spots were sealed with a SecureSealTM hybridization chamber (#621503, Grace Bio-Labs, Scheme S1). The 2-hour hybridization with fluorescently-labeled target solution took place at a controlled temperature that was below the melting temperature of the double-stranded DNA. The slide was immediately exposed to the home-made UV LED assembly centered at 365 nm (LZ4-00U600, LED Engine). Prior to fluorescent scanning, the hybridization chamber was peeled off by hand and the slide was rinsed at 4 °C with 20 mM phosphate buffered saline solution followed by blowing dry with nitrogen. Fluorescent scanning
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The commercial fluorescent scanner (TyphoonTM FLA 9000, GE Healthcare) was used to excite the slides and to collect fluorescent images. The fluorophore in use was tetramethylrhodamine. The excitation source was a 532-nm laser. The scanning pixel resolution was set at 50 µm.
Results and Discussion We first studied the hybridization of the functionalized azobenzene-modified capture DNA with fluorophore-labeled perfectly-matched targets. Though azobenzene has been reported to quench the fluorescence of various dye molecules,34,35 we did not see marked quenching in this format and were able to measure fluorescence after the hybridization. Figure 1(a) shows post-hybridization fluorescent images of azobenzene-modified DNA slides (“Azo” image on the right) and azobenzene-free DNA slides (“Nonazo” image on the left) with the target concentration between 0.1 nM and 1000 nM. The grey spots correspond to areas that are functionalized with DNA capture sequences. The grey-scale darkness level is proportional to the emitted fluorescence intensity from target sequences that are captured by azobenzenemodified DNA. The “Non-azo” image shows control data for unmodified azobenzene-free DNA. As expected, the fluorescence intensity decreases as the target concentration reduces from 1000 nM to 10 nM. The duplicate spots on the same slide (the 2nd column in the image) show the same trend with only minor variations we attribute to variations in surface properties. Similarly, the “Azo” image— corresponding to the azobenzene-modified DNA slide—shows the expected trend of decreasing fluorescence intensity as the target concentration drops from 1000 nM to 10 nM. For both sequences, under these conditions, the fluorescence is difficult to detect when the target concentration drops to below 1 nM, consistent with literature reports.36,37 Figure 1(b) shows a box and whisker plot of the fluorescence intensity value versus the target concentration over multiple slides, reflecting the distribution of slide-toslide variability associated with our manual spotting. The center line of the box is the median value; the bottom edge of the box is the 3rd quartile; the upper edge of the box is the 1st quartile; the bottom error bar is the minimum value and the upper error bar is the maximum value. Both the slides with azobenzene-
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modified capture sequences, and the azobenzene-free control slides show the expected increase in fluorescence intensity with DNA concentration. Importantly, despite reports of azobenzene being able to quench the emission of nearby fluorophores via pi-pi electronic interactions,34,35 the signal from the fluorescent DNA captured onto the azobenzene-modified spots was almost as intense as that captured on the control slides, with only a small (25% - 30%) decrease in the median fluorescence value for the azobenzene-modified slides being observed at the highest target concentrations. We also note that, in addition to fluorescent quenching, the ~25% - 30% decrease in fluorescent intensity could be attributed to the change in binding efficiency of azobenzene-modified capture sequences. We thus conclude that DNA chips functionalized with azobenzene-modified capture sequences are compatible with commercial fluorescent readout with only minor (
