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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
pubs.acs.org/acschemicalbiology
© 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:
[email protected] 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).
■ REFERENCES(1) Gambari, R. (2004) New trends in the development
oftranscription factor decoy (TFD) pharmacotherapy. Curr.
DrugTargets 5, 419−430.(2) Tomita, N., Ogihara, T., and Morishita,
R. (2003) Transcriptionfactors as molecular targets: Molecular
mechanisms of decoy ODNand their design. Curr. Drug Targets 4,
603−608.(3) Mann, M. J. (2005) Transcription factor decoys: A new
model fordisease intervention. Ann. N. Y. Acad. Sci. 1058,
128−139.(4) Morishita, R., Sugimoto, T., Aoki, M., Kida, I.,
Tomita, N.,Moriguchi, A., Maeda, K., Sawa, Y., Kaneda, Y., Higaki,
J., and Ogihara,T. (1997) In vivo transfection of cis element
″decoy″ against nuclearfactor-κB binding site prevents myocardial
infarction. Nat. Med. 3,894−899.(5) Penolazzi, L., Magri, E.,
Lambertini, E., Calo,̀ G., Cozzani, M.,Siciliani, G., Piva, R., and
Gambari, R. (2006) Local in vivoadministration of a decoy
oligonucleotide targeting NF-κB inducesapoptosis of osteoclasts
after application of orthodontic forces to ratteeth. Int. J. Mol.
Med. 18, 807−811.(6) Metelev, V. G., Kubareva, E. A., and
Oretskaya, T. S. (2013)Regulation of activity of transcription
factor NF-κB by syntheticoligonucleotides. Biochemistry (Moscow)
78, 867−878.(7) Ceo, L. M., and Koh, J. T. (2012) Photocaged DNA
providesnew levels of transcription control. ChemBioChem 13,
511−513.(8) Xie, S., Nie, R., Wang, J., Li, F., and Yuan, W.
(2009)Transcription factor decoys for activator protein-1 (AP-1)
inhibitoxidative stress-induced proliferation and matrix
metalloproteinases inrat cardiac fibroblasts. Transl. Res. 153,
17−23.(9) Souissi, I., Najjar, I., Ah-Koon, L., Schischmanoff, P.
O., Lesage,D., Le Coquil, S., Roger, C., Dusanter-Fourt, I.,
Varin-Blank, N., Cao,A., Metelev, V., Baran-Marszak, F., and
Fagard, R. (2011) A STAT3-decoy oligonucleotide induces cell death
in a human colorectal
carcinoma cell line by blocking nuclear transfer of STAT3 and
STAT3-bound NF-κB. BMC Cell Biol. 12, 14−31.(10) Wang, J., Cheng,
H., Li, X., Lu, W., Wang, K., and Wen, T.(2013) Regulation of
neural stem cell differentiation by transcriptionfactors HNF4−1 and
MAZ-1. Mol. Neurobiol. 47, 228−240.(11) Brieke, C., Rohrbach, F.,
Gottschalk, A., Mayer, G., and Heckel,A. (2012) Light-controlled
tools. Angew. Chem. Int. Ed. 51, 8446−8476.(12) Lee, H. M., Larson,
D. R., and Lawrence, D. S. (2009)Illuminating the chemistry of
life: design, synthesis, and applications of″caged″ and related
photoresponsive compounds. ACS Chem. Biol. 4,409−427.(13) Liu, Q.,
and Deiters, A. (2014) Optochemical control ofdeoxyoligonucleotide
function via a nucleobase-caging approach. Acc.Chem. Res. 47,
45−55.(14) Govan, J. M., Lively, M. O., and Deiters, A.
(2011)Photochemical control of DNA decoy function enables
preciseregulation of nuclear factor κB activity. J. Am. Chem. Soc.
133,13176−13182.(15) The term depurination is used liberally to
denote cleavage of thenucleobase−sugar bond. The
depurination-competent monomers usedin this study are not
technically purines.(16) Lenox, H. J., McCoy, C. P., and Sheppard,
T. L. (2001) Site-specific generation of deoxyribonolactone lesions
in DNA oligonu-cleotides. Org. Lett. 3, 2415−2418.(17) Trzupek, J.
D., and Sheppard, T. L. (2005) Photochemicalgeneration of ribose
abasic sites in RNA oligonucleotides. Org. Lett. 7,1493−1496.(18)
Wang, Y., Sheppard, T. L., Tornaletti, S., Maeda, L. S.,
andHanawalt, P. C. (2006) Transcriptional inhibition by an
oxidizedabasic site in DNA. Chem. Res. Toxicol. 19, 234−241.(19)
Kotera, M., Bourdat, A.-G., Defrancq, E., and Lhomme, J.(1998) A
highly efficient synthesis of oligodeoxyribonucleotidescontaining
the 2′-deoxyribonolactone lesion. J. Am. Chem. Soc.
120,11810−11811.(20) Kotera, M., Roupioz, Y., Defrancq, E.,
Bourdat, A.-G., Garcia, J.,Coulombeau, C., and Lhomme, J. (2000)
The 7-nitroindolenucleoside as a photochemical precursor of
2′-deoxyribonolactone:access to DNA fragments containing this
oxidative abasic lesion. Chem.- Eur. J. 6, 4163−4169.(21) Roupioz,
Y., Lhomme, J., and Kotera, M. (2002) Chemistry ofthe
2-deoxyribonolactone lesion in oligonucleotides: cleavage
kineticsand products analysis. J. Am. Chem. Soc. 124,
9129−9135.(22) Loakes, D., and Brown, D. M. (1994) 5-Nitroindole as
anuniversal base analogue. Nucleic Acids Res. 22, 4039−4043.(23)
Crey-Desbiolles, C., Berthet, N., Kotera, M., and Dumy, P.(2005)
Hybridization properties and enzymatic replication
ofoligonucleotides containing the photocleavable 7-nitroindole
baseanalog. Nucleic Acids Res. 33, 1532−1543.(24) Hayden, M. S.,
and Ghosh, S. (2012) NF-κB, the first quarter-century: remarkable
progress and outstanding questions. Genes Dev.26, 203−234.(25)
Chen, F. E., Huang, D. B., Chen, Y. Q., and Ghosh, G. (1998)Crystal
structure of p50/p65 heterodimer of transcription factor NF-κB
bound to DNA. Nature 391, 410−413.(26) Mergny, J. L., and Lacroix,
L. (2003) Analysis of thermalmelting curves. Oligonucleotides 13,
515−537.(27) Sun, L., and Carpenter, G. (1998) Epidermal growth
factoractivation of NF-κB is mediated through IκBα degradation
andintracellular free calcium. Oncogene 16, 2095−2102.(28) Phelps,
C. B., Sengchanthalangsy, L. L., Malek, S., and Ghosh,G. (2000)
Mechanism of κB DNA binding by Rel/NF-κB dimers. J.Biol. Chem. 275,
24392−24399.(29) Lusic, H., Young, D. D., Lively, M. O., and
Deiters, A. (2007)Photochemical DNA activation. Org. Lett. 9,
1903−1906.(30) Caruthers, M. H. (1991) Chemical synthesis of DNA
and DNAanaloguesa. Acc. Chem. Res. 24, 278−284.
ACS Chemical Biology Articles
DOI: 10.1021/acschembio.6b00130ACS Chem. Biol. 2016, 11,
1631−1638
1637
http://pubs.acs.orghttp://pubs.acs.org/doi/abs/10.1021/acschembio.6b00130http://pubs.acs.org/doi/abs/10.1021/acschembio.6b00130http://pubs.acs.org/doi/suppl/10.1021/acschembio.6b00130/suppl_file/cb6b00130_si_001.pdfmailto:[email protected]://dx.doi.org/10.1021/acschembio.6b00130
-
(31) Heckel, A. (2007) Nucleobase-caged phosphoramidites
foroligonucleotide synthesis. Curr. Protoc. Nucleic Acid Chem.,
1.17.1−1.17.26.(32) Chenoweth, D. M., Harki, D. A., Phillips, J.
W., Dose, C., andDervan, P. B. (2009) Cyclic pyrrole-imidazole
polyamides targeted tothe androgen response element. J. Am. Chem.
Soc. 131, 7182−7188.(33) Yang, B., Chang, Y., Weyers, A. M.,
Sterner, E., and Linhardt, R.J. (2012) Disaccharide analysis of
glycosaminoglycan mixtures byultra-high-performance liquid
chromatography-mass spectrometry. J.Chromatogr. 1225, 91−98.(34)
Kuhn, H. J., Braslavsky, S. E., and Schmidt, R. (2004)
Chemicalactinometry. Pure Appl. Chem. 76, 2105−2146.(35) Demas, J.
N., Bowman, W. D., Zalewski, E. F., and Velapoldi, R.A. (1981)
Determination of the quantum yield of the ferrioxalateactinometer
with electrically calibrated radiometers. J. Phys. Chem.
85,2766−2771.(36) Zhu, Y., Pavlos, C. M., Toscano, J. P., and Dore,
T. M. (2006) 8-Bromo-7-hydroxyquinoline as a photoremovable
protecting group forphysiological use: Mechanism and scope. J. Am.
Chem. Soc. 128, 4267−4276.(37) Chenoweth, D. M., Poposki, J. A.,
Marques, M. A., and Dervan,P. B. (2007) Programmable oligomers
targeting 5′-GGGG-3′ in theminor groove of DNA and NF-κB binding
inhibition. Bioorg. Med.Chem. 15, 759−770.(38) Hexum, J. K.,
Tello-Aburto, R., Struntz, N. B., Harned, A. M.,and Harki, D. A.
(2012) Bicyclic cyclohexenones as inhibitors of NF-κB signaling.
ACS Med. Chem. Lett. 3, 459−464.
ACS Chemical Biology Articles
DOI: 10.1021/acschembio.6b00130ACS Chem. Biol. 2016, 11,
1631−1638
1638
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