Catch and Release DNA Decoys: Capture and PhotochemicalDissociation of NF-κB Transcription FactorsNicholas B. Struntz and Daniel A. Harki*

Department of Medicinal Chemistry, University of Minnesota, 2231 6th Street S.E., Minneapolis, Minnesota 55455, United States

*S Supporting Information

ABSTRACT: Catch and release DNA decoys (CRDDs) are anew class of non-natural DNA probes that capture anddissociate from DNA-binding proteins using a light trigger.Photolytic cleavage of non-natural nucleobases in the CRDDyields abasic sites and truncation products that lower theaffinity of the CRDD for its protein target. Herein, wedemonstrate the ability of the first-generation CRDD to bindand release NF-κB proteins. This platform technology shouldbe applicable to other DNA-binding proteins by modificationof the target sequence.

DNA decoys modulate cellular and viral transcription bysequestering regulatory proteins, both activators andrepressors, from their endogenous DNA binding sites, therebyaltering gene expression.1−3 Transcription factors (TFs) such asNF-κB,4−7 AP-1,8 STAT3,9 and HNF4-1/MAZ-110 have beensuccessfully targeted by sequence-specific DNA decoys, andthese reagents have demonstrated utility in both cell cultureand animal models of disease. Consequently, DNA decoysconstitute powerful tool compounds for mechanistic biochem-ical studies and promising therapeutic agents for regulatingaberrant TF activities.The utilization of light as a bio-orthogonal trigger to regulate

the activities of biologically active molecules has garneredsubstantial attention in recent years.11,12 Incorporation ofnitrobenzyl derivatives onto nucleobases of DNA and RNAoligonucleotides, which sterically block binding to cognateproteins or nucleic acids, has yielded an arsenal of cagedreagents with broad utilities in transcriptional and translationalregulation.7,13 A caged DNA decoy targeting NF-κB proteinshas been developed that enables light-mediated spatiotemporalcontrol of expression of NF-κB-target genes.14 This “off-to-on”DNA decoy is activated by photolysis of the appended caginggroups, which reveals an exogenous DNA hairpin thatsequesters NF-κB proteins.We report a novel class of DNA decoys, termed catch and

release DNA decoys (CRDDs), that capture and release DNA-binding proteins using a light trigger (Figure 1a). Unlike cagedDNA decoys, CRDDs are an “on-to-off” platform that utilizeslight to promote photochemical destruction of the DNA decoyand dissociation of the protein−DNA complex. Our designutilizes depurination-competent15 mimics of natural nucleo-bases that when incorporated into a DNA decoy function asnatural nucleobases and enable decoy binding to its designedprotein target. However, CRDD photolysis results in formationof multiple abasic sites within the decoy, as well as truncateddecoys resulting from β- and δ-elimination at abasic sites,

yielding a modified CRDD that possesses significantlydiminished affinity for its design protein target. Consequently,CRDD photolysis enables release of the sequestered proteintarget. Here, we report our first-generation CRDD that cancapture and release NF-κB proteins.

■ RESULTS AND DISCUSSIONDesign of CRDDs Targeting NF-κB TFs. Incorporation of

2-nitrobenzylethers in place of native nucleobases in DNAoligonucleotides has been utilized as a strategy to generateabasic sites photochemically with sequence specificity.16−18

Additionally, 7-nitroindole (7-NI; 1) has been shown tophotochemically depurinate in DNA oligonucleotides, yieldinga 2′-deoxyribolactone (2) abasic site in DNA (mechanism:Figure S1), which can undergo β- and δ-elimination, resultingin strand cleavage, and a 7-nitrosoindole (3) byproduct (Figure1b).19−21 Given the obvious structural similarities betweenindole heterocycles and purine nucleobases, which is furtherreinforced by work demonstrating that 5-nitroindole can serveas a “universal base” in DNA22 and that both 5- and 7-NInucleobases can be enzymatically recognized by Klenowfragment DNA polymerase I,23 we hypothesized that 7-NImay suitably mimic natural purines in established DNA decoysthat bind NF-κB proteins. Furthermore, we hypothesized thatphotolysis of the NF-κB−decoy complex would enable proteinrelease through photochemical destruction of the decoy aspreviously described (Figure 1c).The NF-κB signaling pathway regulates scores of cellular

processes associated with inflammation, cell survival, andproliferation, and aberrant NF-κB activity is frequently foundin cancer, cardiovascular disease, and autoimmune diseases.24

Received: February 9, 2016Accepted: March 22, 2016Published: April 7, 2016

Articles

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© 2016 American Chemical Society 1631 DOI: 10.1021/acschembio.6b00130ACS Chem. Biol. 2016, 11, 1631−1638

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Given the fundamental role of NF-κB signaling in many humandiseases, as well as strong precedence for the development ofNF-κB-targeted DNA decoys,4−7 including caged reagents,14

we developed our first CRDD against NF-κB proteins. The fourpurine site of the NF-κB consensus sequence (5′-GGGRN-YYYCC-3′, where R is a purine, Y is a pyrimidine, and N is anynucleotide) is crucial for DNA binding of p50.25 Therefore, weutilized established NF-κB decoy 414 as our base hairpinsequence for optimizing NF-κB-targeted CRDDs (Figure 2a).One-to-three purines included in or flanking the 4-G site werereplaced with 7-NI nucleotides, yielding CRDDs 5, 7, and 9.The substitutions of the 7-NIs were also placed in succession toamplify the result of multiple depurination events in aconcentrated area. These decoys were synthesized using theknown 7-NI phosphoramidite20 under standard solid-phaseDNA synthesis conditions. Photolysis of 5, 7, and 9 followed bypurification afforded decoys 6, 8, and 10 containing one, two,and three abasic sites, respectively. A control scrambled DNAdecoy 11, a scrambled CRDD 12 containing three 7-NInucleotides, and DNA decoy 13 with three base pairmismatches in place of the three 7-NI nucleotides were alsosynthesized.Thermal Stability of DNA Decoys. The thermal stability

of DNA duplexes and hairpins containing the 7-NI nucleobasewas studied by UV thermal melting experiments.26 First, 7-NIwas singly incorporated into oligonucleotide 20 and thermalmelting of duplex DNA containing all natural nucleotideshybridized to 7-NI was measured. In addition, the DNAstability of duplexes containing guanine and 5-NI nucleobaseswas included for comparison (Table S1).22,26 In comparison toG−C pairing, incorporation of 5-NI into duplex DNA in place

of guanine decreases stability (ΔTm = −7.5 °C), whereas 7-NIis more destabilizing (ΔTm = −15.2 °C). Thermal melting ofCRDDs 5, 7, and 9 and the stability of their correspondingabasic photoproducts 6, 8, and 10 were then evaluated (Figure2b). The introduction of a single 7-NI nucleotide into CCRD 5decreased duplex stability as expected (ΔTm = −4.1 °C), butphotochemical introduction of the abasic site, yielding 6,increased stability compared to that of 5 (ΔΔTm = +2.6 °C).From this result, it was hypothesized that more than one abasicsite would be needed to sufficiently disrupt duplex formationand overall binding affinity after depurination. The thermalstabilities of 7 and 9 were lower in comparison to that ofnonmodified decoy 4 (ΔTm = −10.3 and −12.2 °C,respectively), yet both are sufficiently stable (Tm = 72.3 and70.4 °C, respectively). As anticipated, photochemical introduc-tion of multiple abasic sites, forming 8 (two abasic sites), isdestabilizing to duplex DNA (ΔTm = −12.3 °C compared tothat of 4; ΔTm = −2.0 °C compared to that of 7) and evengreater for 10 (ΔTm = −15.8 °C compared to that of 4; ΔTm =−3.6 °C compared to that of 9), which contains three abasicsites. The thermal stability of scramble 12 containing three 7-NIs was lower in comparison to that of nonmodified scrambledecoy 11 (ΔTm = −12.4 °C), which is similar to that previouslyseen with CRDD 9 in comparison to 4. The DNA decoy withthree base pair mismatches 13 in place of the three 7-NI

Figure 1. Catch and release DNA decoy (CRDD) targeting NF-κBtranscription factors. (a) Photolysis of DNA decoys containingphotoresponsive nucleotides (X with stars) with UV light (hν) resultsin the formation of abasic sites (_) and strand cleavage products. (b)7-Nitroindole-containing oligonucleotides (1) depurinate with UVlight, resulting in the formation of 2′-deoxyribolactone (2) and 7-nitrosoindole (3) products. (c) Incorporation of three 7-nitroindolenucleotides (X = 1) into a DNA decoy sequence known to target NF-κB proteins still permits protein binding (catch). Photolysis of thedecoy with UV light (350 nm) results in the formation of multipleabasic sites and truncation products that have lower affinity for theprotein, thereby enabling dissociation of the NF-κB−CRDD complex(release).

Figure 2. Thermal stability of synthesized NF-κB-directed (4−10),scramble (11 and 12), and three base pair mismatch (13) DNAdecoys. (a) Synthesized decoys 4−13 (X = 1; _ = 2). (b) Thermalmelting of 4−13 demonstrating DNA duplex destabilization resultingfrom incorporation of 7-nitroindoles (CRDDs 5, 7, 9, and 12) and,more predominantly, abasic sites (CRDDs 8 and 10). Thermal meltingof 13 demonstrates destabilization due to three base pair mismatches.Thermal melting experiments were performed in 10 mM sodiumcacodylate, 10 mM KCl, 10 mM MgCl2, 5 mM CaCl2, pH 7.0 buffer.ΔTm values are relative to 4, except 12, which is relative to 11. Mean ±SD (n = 4).

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nucleotides also displayed a lowered Tm compared to that ofnative 4 (ΔTm = −10.4 °C).Electrophoretic Mobility Shift Assays of NF-κB−DNA

Complexes. We next evaluated the ability of the decoys tocapture NF-κB proteins by electrophoretic mobility shift assays(EMSAs). 32P-end-labeled 4−13 were added to a solution ofrecombinant p50−p65 proteins, and protein binding wasmeasured. The NF-κB-directed DNA decoys exhibited specificbinding toward NF-κB proteins, as little observable bindingoccurred with scramble and base pair mismatch decoys 11−13(Figure S2). To characterize the NF-κB complexes responsiblefor binding to DNA decoys, supershift EMSAs were carried out(Figure S3). The p50 utilized in all experiments is a partiallytruncated recombinant protein (35−381; 433 amino acids forwild-type enzyme). The p65 recombinant protein utilized inthese experiments is primarily the DNA-binding domain withan N-terminal GST tag (1−306; 551 amino acids for wild-typeenzyme). Addition of p50 antibody to NF-κB−4 complexescompletely supershifted the band, indicating that p50 protein ispresent in all complexes bound to 4 (Figure S3a). However,addition of p65 antibody only partially supershifted the NF-κBcomplex (Figure S3a). These data suggest that the observedNF-κB−4 complex is composed of both p50−p65 heterodimers(supershift with p65 antibody) and p50−p50 homodimers (nosupershift with p65 antibody), which is consistent with previousstudies.27 The supershift experiment was repeated with a nearfull-length p65 recombinant protein (1−537), which confirmedthese results (Figure S3b). The ability of CRDD 9 (containingthree 7-NIs) to bind the NF-κB complexes was similarlyconfirmed by EMSA supershift analysis (Figure S3c). Tosupport these EMSA results, we performed quantitative EMSAtitrations with NF-κB proteins to obtain equilibrium dissoci-ation constants for all three possible NF-κB complexes withnative DNA decoy 4 (Figure 3). The p50−p65 heterodimer

demonstrated the highest binding affinity (Kd = 15.3 nM). Thep50 and p65 homodimer proteins were also measured (Kd =31.1 and 91.7 nM, respectively), revealing that the p65homodimer has a 6-fold lower binding affinity for 4 than thep50−p65 heterodimer, which is consistent with a previousstudy.28 Therefore, our studies of CRDD binding to NF-κBproteins are sampling a mixture of both p50 and p65heterodimers and homodimers.As shown in Figure 4a, CRDDs 5, 7, and 9 demonstrated the

ability to bind the NF-κB complex similar to that of nativedecoy 4. Quantitative analysis using densitometry revealed an

approximately 50% decrease in the signal of CRDD 9 comparedto that of native 4. Decoys 6 and 8, however, retain the abilityto bind despite containing one and two abasic sites. Decoy 10,containing three abasic sites, demonstrated no observablecomplex formation upon addition of proteins. This resultrevealed that the photochemical transformation of 9 to 10abolishes NF-κB binding; therefore, we utilized this compoundfor further studies.To corroborate our EMSA results, we performed quantitative

EMSA titrations with NF-κB proteins and 4, 9, and 10 toobtain equilibrium dissociation constants for our probes. DNAdecoy 4 (Kd = 15.3 nM) and CRDD 9 (Kd = 36.3 nM)exhibited comparable binding affinities for NF-κB proteins(Figure 4b). Remarkably, CRDD 10, which contains threeabasic sites in the p50 recognition domain, exhibited noobservable binding to the NF-κB proteins (Kd > 500 nM).These data further support our model by which CRDDdepurination ablates protein recognition.

Characterization of CRDD Photoproducts. To charac-terize the kinetics and the identities of the photoproductsresulting from irradiation of 9, we utilized liquid chromatog-raphy−mass spectrometry (LC-MS) analysis of an irradiatedaqueous sample. As shown in Figure 4c, irradiation of 9 yieldedfast photolysis (t1/2 = 1.0 min; 350 nm light; light intensity:5.66 × 10−8 ein cm−2 s−1) whereby 90% of 9 was converted tophotoproducts within 4.5 min. The quantum yield (Φ) of 9 wasdetermined to be 0.0104, which is comparable, albeit lower,than that of 6-nitropiperonyloxymethyl (NPOM)-protectedthymine (Φ = 0.094).29 After 25 min of irradiation of CRDD 9,very little decoy remained and multiple abasic decoys andtruncation products resulting from β- and δ-elimination weredetected (Figure 4d). As expected, formation of decoys withone abasic site increases immediately, peaks around 5 min, anddecreases as multiple abasic sites are formed. Decoys with one,two, and three abasic sites (14, 15, and 10, respectively) wereobserved after 25 min of irradiation. These abasic decoys resultin approximately 65% of the total number of photoproductsformed (Figure 4d, dashed lines). Several truncated products(16−19) are formed by β- and δ-eliminations of the 3′ and 5′phosphates because of the instability of the abasic lactoneswithin DNA.21 Of these truncated products, 2−6 nucleotidesare cleaved from the 5′ end of the decoy (Figure 4d, solidlines). Consequently, photolysis of 9 results in substantialmodification to the essential 4-G NF-κB binding site, whichdisrupts protein−CRDD binding.

Catch and Release of NF-κB Proteins. To assess theability of CRDD 9 to release the NF-κB complex photochemi-cally, a solution of 32P-labeled 9 and recombinant proteins wasincubated in binding buffer, followed by treatment with 350 nmlight. Photolysis of the 9-NF-κB complex, over time, drives therelease of the TFs due to transformations to the DNA decoy toyield abasic sites and truncation products (Figure 5a). After 4min of irradiation of the 9−NF-κB complex, approximately 50%of the NF-κB proteins are released even though the protein is inlarge excess (Figure 5b). Recovery of the complex can beobtained by additional 32P-labeled 9, demonstrating the viabilityof NF-κB proteins to bind DNA after irradiation with light.Additionally, western blot and EMSA analysis of p50 and p65proteins following subjection to the photolysis conditions (350nm light, in H2O) reveal no apparent damage to proteins(Figure S5).

Conclusions. We demonstrate for the first time the captureand photochemical release of TFs using a novel photo-

Figure 3. Quantitative EMSA analysis to measure equilibriumdissociation (Kd) constants for 4 with p50−p65 heterodimer andp50 and p65 homodimer proteins. Recombinant p50 (35−381) andp65 (1−306) proteins were used in this study. Mean ± SD (n = 4).

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responsive CRDD. Photolysis of CRDD 9 resulted in theformation of abasic sites and truncation products, abolishingaffinity for binding to NF-κB proteins. These results have

demonstrated that CRDDs can be used to effectively captureand release TFs, which complement existing caged DNAdecoys. The utilization of this technology in cell culture, as well

Figure 4. (a) EMSAs with 4−10. 5′-32P-labeled 4−10 incubated with p50 (35−381) and p65 (1−306) recombinant proteins display formation ofthe NF-κB−CRDD complexes. NF-κB binding is observed until three abasic sites are formed (i.e., 10). Scrambled decoys 11 and 12 and mismatchdecoy 13 demonstrate no observable complex formation (Figure S2). (b) Quantitative EMSA analysis to measure equilibrium dissociation (Kd)constants for 4, 9, and 10 with recombinant p50 (35−381) and p65 (1−306) proteins. (c, d) Photolysis of CRDD 9 (350 nm light). Samples wereanalyzed by ion-extracted LC-MS. (c) Photolytic decay curve of 9 with calculated half-life and quantum yield (R2 = 0.97). (d) Formation of abasicsites and truncation products 10 and 14−19 resulting from photolysis of 9 (P = 5′-phosphate; X = 1). Dashed line denotes full-length DNA decoyscontaining abasic products, and solid lines denote truncation products (some contain abasic sites as well). Isomers denote constitution isomersresulting from photolysis of 9 (e.g., X and _ are in different arrangements). Mean ± SD (n = 4). An LC-MS chromatogram of the photoproductsfrom irradiation of 9 can be found in Figure S4.

Figure 5. Quantitative EMSA with CRDD 9. (a) 5′-32P-labeled 9 incubated with p50 (35−381) and p65 (1−306) recombinant proteins yieldscomplex formation (catch, t = 0 min), which is dissociated upon photolysis with 350 nm light (release) in a time-dependent manner. Recovery of theNF-κB−9 complex can be obtained by addition of 32P-labeled 9. (b) Densitometry of EMSAs yielding the half-life of NF-κB release (R2 = 0.93).Mean ± SD (n = 3).

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as the development of second-generation depurinationmonomers and new applications, will be reported in due course.

■ METHODSSolid-Phase DNA Synthesis. Oligonucleotides were synthesized

using standard solid-phase phosphoramidite chemistry on an AppliedBiosystems 394 DNA/RNA synthesizer.30 7-NI phosphoramidite wassynthesized as previously described.20,31 All other phosphoramidites,reagents, and solid supports (1.0 μmol) were purchased from GlenResearch Corporation. 7-NI was incorporated into the oligonucleotideby manual coupling, whereas all other nucleotides were incorporatedwith automated coupling. Automated DNA synthesis was pausedimmediately prior to incorporation of 7-NI, and the solid support wasremoved from the synthesizer. To achieve manual coupling, 7-NIphosphoramidite (15 mg) was dissolved in anhydrous MeCN (200μL), loaded into a syringe (1 mL), and attached to one side of thesolid support. A second syringe (1 mL) was loaded with activator (600μL) and attached to the other end of the solid support. The solutionswere then mixed through the solid support vessel manually using thetwo syringes for 20 min. Afterward, the solid support vessel wasdrained, washed with anhydrous MeCN (1 mL), and returned to thesynthesizer. This procedure for manual coupling was performed for all7-NI incorporations. Following synthesis, the resin was transferred to afritted reaction vessel. Concentrated aqueous ammonium hydroxide(2.5 mL) was added, and the vessel was placed in a shaker for 18 h atrt. After deprotection, the solution was filtered into a centrifuge tube(10 mL) and distilled water (2 mL) was added. The ammoniumhydroxide was evaporated in vacuo (samples were transferred tomicrocentrifuge tubes and placed in a SpeedVac), and the remainingsolution was purified by HPLC (see below). After purification, theoligonucleotides were desalted with DNase/RNase-free H2O usingIllustra NAP-5 columns (Sephadex G-25 DNA grade, GE Healthcare)according to the manufacturer’s instructions. The desalted oligonu-cleotides were quantified by UV−vis spectroscopy (A260, usingpredicted molar extinction coefficients for native dNTPs and ε =5900 M−1 cm−1 for 7-NI at 260 nm) and confirmed by LC-MS (seebelow). The purity was assessed by HPLC reinjection of the purifiedoligonucleotides (see Supporting Information for chromatograms).Oligonucleotide 4. Purity = 95.9% (260 nm). MS calcd, 9502.2;

found, 9501.6 (parent).Oligonucleotide 5. Purity = 97.9% (260 nm). MS calcd, 9513.2;

found, 9513.9 (parent).Oligonucleotide 6. Purity = 95.8% (260 nm). MS calcd, 9367.1;

found, 9368.1 (parent).Oligonucleotide 7. Purity = 98.6% (260 nm). MS calcd, 9524.2;

found, 9524.7 (parent).Oligonucleotide 8. Purity = 98.4% (260 nm). MS calcd, 9232.9;

found, 9233.1 (parent).Oligonucleotide 9. Purity = 98.5% (260 nm). MS calcd, 9551.2;

found, 9551.7 (parent).Oligonucleotide 10. Purity = 96.1% (260 nm). MS calcd, 9114.8;

found, 9113.5 (parent).Oligonucleotide 11. Purity = 93.0% (260 nm). MS calcd, 9502.2;

found, 9502.0 (parent).Oligonucleotide 12. Purity = 95.3% (260 nm). MS calcd, 9551.2;

found, 9550.8 (parent).Oligonucleotide 13. Purity = 94.5% (260 nm). MS calcd, 9486.2;

found, 9486.4 (parent).Oligonucleotide 20. Purity = 93.0% (260 nm). MS calcd, 4557.0;

found, 4556.8 (parent).Oligonucleotides shown in Supplemental Table 1, except for 20,

were purchased HPLC-purified from Integrated DNA Technologies.HPLC Purification and LC-MS Analysis. Oligonucleotides were

HPLC purified on an Agilent 1200 series instrument equipped with adiode array detector and a PLRP-S column (8 μm, 100 Å, 4.6 × 150mm, Agilent Technologies). The analysis method (2.750 mL/min flowrate) involved isocratic 100 mM TEAA (aqueous, pH 7.0, Sigma-Aldrich; 0−5 min) followed by a linear gradient to 10% 100 mMTEAA/MeCN (1:1, 5−10 min) and finally a linear gradient of 30−

70% 100 mM TEAA/MeCN (1:1, 10−45 min). Wavelengthsmonitored = 215 and 260 nm. LC-MS was performed on an Agilent1100 series HPLC instrument equipped with an Agilent MSD SL iontrap mass spectrometer (operating in negative ion mode). A ZorbaxSB-C18 column (5 μm, 300 Å, 0.5 × 150 mm, Agilent Technologies)was used for LC-MS analysis. The analysis method (15 μL/min flowrate) involved 15 mM aqueous NH4OAc containing 2% MeCNfollowed by a linear gradient of 2−25% MeCN (0−15 min) and 25−60% MeCN (15−25 min). Wavelengths monitored = 215 and 260 nm.

Thermal Melting Analysis. Thermal melting analyses wereperformed on a temperature-controlled Agilent Cary 100 UV−visspectrophotometer containing a six-cell block with a path length of 1cm. A degassed aqueous solution of 10 mM sodium cacodylate, 10mM KCl, 10 mM MgCl2, and 5 mM CaCl2 (pH 7.0) was used asanalysis buffer.32 Oligonucleotides (1 nmol) were mixed in the buffer(1 mL). Before data collection, samples were heated at 90 °C andcooled to a starting temperature of 30 °C with a 5 °C/min ramp. Datapoints were recorded at λ = 260 nm every 12 s on a 0.5 °C/min rampfrom 30 to 90 °C. After data collection, the sample was cooled to 30°C with a 5 °C/min ramp. The method was repeated to obtain atechnical replicate. The experiment was repeated to obtain a biologicalreplicate (n = 4 total analyses). The reported thermal meltingtemperatures (Tm) were calculated from the maximum of the firstderivative of the denaturation curve (Cary WinUV ThermalApplication, v 4.20). Mean Tm values (with standard deviation) werecalculated from the individual Tm values obtained from each replicate(n = 4).

Photolysis, Exponential Decay, and Quantum Yield Analysis.DNA photolysis experiments were carried out using a Rayonetphotochemical reactor (RMR-600, Southern New England UltravioletCo.) fitted with eight 350 nm bulbs. To enable quantitative analysis ofphotochemical decay of DNA decoys, calibration plots for each DNAdecoy were generated. Increasing concentrations of each DNA decoy(0.55, 1.65, 4.94, 14.81, 44.44, 133.33 pmol) were added to a fixedconcentration of a nonmodified DNA oligonucleotide (5′-TAACTA-3′, 100 pmol) and analyzed by extracted ion current LC-MS (masseswere monitored at the −9 charge state for decoys).33 A calibration plotwas created by plotting the ratio of decoy/standard area under thecurve versus DNA decoy concentration, yielding calibration plots witha slope-intercept equation of R2 > 0.99.

Quantitative analysis of DNA decoy photolysis was performed bydissolving the DNA decoy (800 pmol) in DNase/RNase-free H2O andthen adding the solution to conical pulled point vial inserts (250 μL;Agilent, 8010-0125). Vessels containing the aqueous DNA solutionwere placed into the photochemical reactor and irradiated (lightintensity: 5.66 × 10−8 ein cm−2 s−1, calculated as described below).Aliquots (4 μL) were taken at several time points (1, 2, 5, 7, 10, 15, 20,25 min), diluted with standard (1 μL), and then analyzed by LC-MS.The concentration of the decoy species from irradiation wasdetermined by fitting the decoy/standard ratios from each sampleinto the slope-intercept equation from the calibration plot to yield theamount of decoy (pmol) in the sample. This process was repeated foreach prominent molecular ion observed in the photolysis sample.Furthermore, this quantitative analysis method assures comparableionization properties for the photolyzed products in comparison to thenonirradiated sample. First-order decay analysis (GraphPad Prism,v5.0b) was performed with the data (percentage of starting materialover time) to obtain the half-life (t1/2) of the DNA decoy. Mean t1/2values (with standard deviation) were calculated from the fitting of thedecay curve with the individual data points obtained from eachreplicate (n = 4).

Quantum yield (Φ) calculations were carried out to determine theefficiency of photolysis of CRDD 9 (eq 1). The intensity of the lightsource (I, eq 2) was determined using K3[Fe(C2O4)3] actinometry aspreviously described.34,35 In brief, a solution of K3[Fe(C2O4)3]·3H2Oin distilled H2O (6 M, 2 mL) was irradiated for 180 s in the Rayonetequipped with eight 350 nm bulbs. After irradiation, the sample wastransferred to a volumetric flask (25 mL). To the flask were addedaqueous buffer (3 mL; recipe to make a 500 mL solution of aqueousbuffer: 300 mL of 1.0 M NaOAc, 180 mL of 1.0 M H2SO4, and 20 mL

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distilled H2O), phenanthroline solution (3 mL of 0.1% v/vphenanthroline in distilled H2O), KF solution (1 mL of a 2.0 Msolution), and distilled water (∼18 mL, to 25 mL). The solution wasplaced in the dark for 1 h. A nonirradiated sample was prepared in thesame manner. After 1 h, the solutions were transferred to a cuvette andA510 was measured for both samples. The Rayonet light intensity wasthen calculated using eq 2 (5.66 × 10−8 ein cm−2 s−1). The extinctioncoefficient at 350 nm (ε350) of CRDD 9 was calculated by UV−visabsorbance using the Beer−Lambert law (7070 M−1 cm−1). Theirradiation time for 90% conversion (t90%) of the DNA decoy wascalculated from the first-order decay equation (above). Quantum yieldwas then calculated using eq 1 to give a value less than 1.36

σΦ = × × −I t( )90%1

(1)

where σ (cm−2 mol−1) is equal to 1000 × ε350 of the DNA decoy

ε=

× × Δ× × × Φ ×

− −IV V A

V t(ein cm s )

1000 (mL/l)2 1 1 3 510

510 2 Fe (2)

where V1 is the volume of K3[Fe(C2O4)3] irradiated (mL), V2 is thevolume of the K3[Fe(C2O4)3] solution transferred to the volumetricflask (mL), V3 is the volume of the volumetric flask (mL), ΔA510 is thedifference in the absorbance at 510 nm between the irradiated andnonirradiated samples, ε510 is the extinction coefficient of K3[Fe-(C2O4)3] at 510 nm (11 100 M

−1 cm−1),34 ΦFe is the quantum yield ofK3[Fe(C2O4)3]·3H2O (1.21),

34 and t is the time irradiated (s).32P-DNA Radiolabeling. DNA decoys in DNase/RNase-free

water were annealed by heating at 95 °C in a heating block for 5min, followed by slow cooling to rt. To a microcentrifuge tube (1.7mL) were added the annealed DNA decoy (50 pmol) and T4polynucleotide kinase (PNK) buffer (5 μL of a 10× solution, ThermoScientific). DNase/RNase-free H2O was added to yield a final volumeof 40 μL. The reaction tube was placed into a shielded rack, and then[γ-32P]-ATP (5 μL; 6000 Ci/mmol, PerkinElmer) was added. PNKwas diluted in DNase/RNase-free H2O (1:10) and was then added tothe reaction (5 μL). The reaction was briefly mixed, centrifuged (toremove any material from cap), and then placed in a 37 °C heat blockfor 30 min. Heating at 70 °C for 30 min in the second heat block wasthen used to inactivate the kinase. The radioactive reaction mixturewas transferred to an Illustra MicroSpin G-50 column (GE Healthcare,prepared according to the vendor’s instructions) and centrifuged at1500 rpm for 20 s to yield 32P-labeled oligonucleotides. Theradioactivity of the oligonucleotides was quantified (counts/min/μL)by transferring an aliquot to an Eppendorf tube followed by analysison a Beckman LS 6500 multipurpose scintillation counter (drycounting).Electrophoretic Mobility Shift Assays. Binding reactions

containing binding buffer (2 μL of a 10× solution; 10× solution:100 mM Tris, 10 mM EDTA, 500 mM NaCl, and 10% NP-40),37

recombinant p50 protein (0.5 μL; 0.50 μg/μL, Enzo Life Sciences,BML-UW9885-0050, amino acids 35−381), recombinant p65 protein(0.5 μL; 0.50 μg/μL Sino Biological, 12054-H09E, amino acids 1−306), and DNase/RNase-free H2O (to a final volume of 20 μL) wereprepared in microcentrifuge tubes (0.65 mL) and incubated on ice for30 min. 32P-labeled DNA decoys (1 μL, 25 000 counts/min/μL) wereadded to the binding reaction and then incubated at 37 °C for 10 min.For supershift experiments, binding reactions containing recombi-

nant p50 proteins (0.5 μL; 0.50 μg/μL, Enzo Life Sciences, BML-UW9885-0050, amino acids 35−381), recombinant p65 protein (0.5μL; 0.50 μg/μL Sino Biological, 12054-H09E, amino acids 1−306[Figures S3a and S3c] or 2.5 μL; 0.10 μg/μL Active Motif, 31302,amino acids 1−537 [Figure S3b]), p50 antibody (10 μL; 200 μg/0.1mL, Santa Cruz Biotechnology, sc-7178x), or p65 antibody (10 μL;200 μg/0.1 mL, Santa Cruz Biotechnology, sc-8008x) were prepared inmicrocentrifuge tubes (0.65 mL) and incubated on ice for 60 min. 32P-labeled DNA decoys (1 μL, 25 000 counts/min/μL) were added tothe binding reaction and then incubated at 37 °C for 10 min.For binding constant studies (Figures 3 and 4b), the DNA

concentration was held constant (20 000 counts/min/μL) and titratedwith increasing p50−p65 heterodimer, p50−p50 homodimer, or p65−

p65 homodimer at various concentrations (0, 0.90, 9.00, 17.98, 26.98,35.97, 44.96, 89.92, 134.88, and 179.84 nM; 500 nM was used only fordecoy 10) similar to that previously described.28 Recombinant p50protein was from Enzo Life Sciences (BML-UW9885-0050, aminoacids 35−381), and recombinant p65 protein was from Sino Biological(12054-H09E, amino acids 1−306). The fraction of DNA bound ineach reaction was determined by dividing the densitometry of eachbound band by the total densitometry of the bound and free bands.These fractions were then plotted on a semilogarithmic plot(GraphPad Prism, v5.0b), and the equilibrium dissociation constantswere calculated. Mean ± SD (n = 3).

For catch and release, binding reactions were then transferred toglass HPLC vial inserts (each 20 μL binding reaction was pipetted intoindividual inserts) and irradiated (with the exception of nonirradiatedcontrol samples) in the Rayonet with eight 350 nm bulbs (5.66 × 10−8

ein cm−2 s−1) at rt. The samples were taken out of the Rayonet atvarious time points and transferred (∼20 μL volume) to a newmicrocentrifuge tube (0.65 mL). For rebinding studies (Figure 5a, lane10), additional 32P-labeled DNA was added to only that sample (1 μL,25 000 counts/min/μL of DNA). All samples and controls were thenincubated at 37 °C for 2 h.

For photochemical stability studies, binding reactions containingbinding buffer, recombinant p50 protein (0.5 μL; 0.50 μg/μL, EnzoLife Sciences, BML-UW9885-0050, amino acids 35−381), recombi-nant p65 protein (0.5 μL; 0.50 μg/μL Sino Biological, 12054-H09E,amino acids 1−306), and DNase/RNase-free H2O (to a final volumeof 19 μL) were prepared in microcentrifuge tubes (0.65 mL) andincubated at rt for 10 min. The sample to be irradiated (19 μL) waspipetted into a glass HPLC vial insert and irradiated in the Rayonetwith eight 350 nm bulbs (5.66 × 10−8 ein cm−2 s−1) at rt for 1 h. Thesample was then transferred to a new microcentrifuge tube. 32P-labeledDNA decoys (1 μL, 25 000 counts/min/μL) were added to thebinding reaction and then incubated at 37 °C for 10 min.

For gel analysis, loading dye (2 μL, 10× solution; 0.5× TBE, 40%glycerol, 2 mg/mL Orange G dye, Sigma) was added to each reactionand samples were loaded onto a 5% nondenaturing PAGE gel that wasprerun at 200 V for 1 h in 0.5× TBE. Samples were electrophoresed at200 V until the loading dye was ∼3/4 down the gel. The gel wastransferred to filter paper (Bio-Rad; the plates were pried apart and thegel was placed on the wetted filter paper), covered with plastic wrapand cellophane (Bio-Rad), and dried for 1 h (Gel Air Dryer, Bio-Rad).The gel was transferred to a phosphorimager screen overnight andthen analyzed on a Typhoon FLA 7000 biomolecular imager (GEHealthcare). Images were analyzed using ImageQuant TL software (v7.0, GE Healthcare).

Western Blots. Binding reactions were prepared as describedabove for EMSA analysis except that the binding buffer was omitted.Western blots were performed as previously described.38 The sampleto be irradiated (20 μL) was pipetted into a glass HPLC vial insert andirradiated in the Rayonet with eight 350 nm bulbs (5.66 × 10−8 eincm−2 s−1) at rt for 1 h. The sample was then transferred to a newmicrocentrifuge tube. To each sample were added NuPAGE 4× LDSsample buffer (5 μL, Invitrogen) and NuPAGE 10× sample reducingagent (2 μL, Invitrogen), and the samples were heated at 99 °C for 5min. Protein samples were separated on a 4−12% SDS-PAGE gradientgel (Invitrogen) using MES SDS running buffer (NuPAGE) and thenelectrotransferred to a poly(vinylidene difluoride) membrane (Im-mobilon). The membrane was transferred to a heat-sealed bagcontaining Odyssey blocking buffer (5 mL, LI-COR Biotech.) to blockthe membrane overnight at 4 °C. Proteins were detected by incubationwith primary antibodies against p65 (5 μL; Santa Cruz Biotechnology,sc-372) and p50 (30 μL; Enzo Life Sciences, ALX-804-043-C100) in aheat-sealed bag containing blocking buffer (5 mL) overnight at 4 °C.The membrane was then briefly washed by gentle rocking in ddH2O(50 mL, 1 min, total 5×) and then incubated with IRDye 800 anti-rabbit (5 μL; LI-COR Biotech., 926-32211) and IRDye 680 anti-mouse (5 μL; LI-COR Biotech., 926-68020) conjugated secondaryantibodies together in a heat-sealed bag containing blocking buffer (5mL) for 2 h at rt. The membrane was again washed via gentle rockingin ddH2O (50 mL, 1 min, total 5×). The immunocomplexes were

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visualized using an Odyssey classic infrared imaging system (LI-CORBiotech.).

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acschem-bio.6b00130.

Mechanism of photolysis of 7-NI nucleotides; EMSAwith 4, 9, 11, 12, and 13; supershift experiments with 4and 9; LC-MS chromatogram of photolyzed 9; westernblots and EMSAs for p50 and p65; LC-MS chromato-grams of synthesized oligonucleotides; and thermalmelting of DNA containing the 7-NI nucleotide (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: daharki@umn.edu.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the University of Minnesota(Academic Health Center, Seed Grant and start-up funds toD.A.H.). N.B.S. acknowledges the American Heart Association(13PRE14640004 and 15PRE22950024) for a predoctoralfellowship and the University of Minnesota graduate school forfunding. We acknowledge the Analytical Biochemistry CoreFacility of the Masonic Cancer Center (University ofMinnesota) for mass spectrometry resources, which issupported by the National Institutes of Health (P30-CA77598).

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