Figure 1.
Selection of ssDNA aptamers targeting DNA-bound cJun.
A) cJun was incubated with the immobilized DNA and the complexes were washed prior to the addition of the ssDNA pool. Six rounds of SELEX were performed with rounds 5 and 6 including a negative selection as illustrated by the dashed arrows. B) Four individual sequences from round 6 of the selection, the conserved motifs are highlighted. C) EMSAs with aptamer-1, 16, 19, and 27. For each aptamer, either cJun/cJun or cJun/cFos was titrated from 0.4 nM to 6.5 nM.
Figure 2.
Secondary structure probing of aptamer-19.
A) 32P-labeled aptamer was digested with S1 nuclease and resolved by denaturing gel electrophoresis. The relative band intensities of the +S1 nuclease lane are plotted according to nucleotide position at the right of the gel. B) The secondary structure of aptamer-19 as predicted by mFold given the single stranded constraints determined by S1 nuclease digestion. Conserved motifs are highlighted in blue. C) Restriction digest with CviKI-1 confirms the presence of the third stem-loop containing the double stranded recognition sequence 5′-AGCC-3′. The CviKI-1 site is labeled in panel B.
Figure 3.
cJun/cJun homodimers, but not cJun/cFos heterodimers make extensive contacts with aptamer-19.
A) Both cJun/cJun and cJun/cFos protect a discrete region surrounding the AP-1 site on a 78nt long dsDNA construct from DNase I digestion. The relative intensities of the digested products are plotted corresponding to nucleotide position. The digestion profile is presented in the absence of protein (solid gray), in the presence of 10 nM cJun/cJun (blue line), or the presence of 10 nM cJun/cFos (red line). B) cJun/cJun broadly protects aptamer-19 from DNase I digestion, while cJun/cFos does not. C) Digestion profile of aptamer-19 upon hydroxyl radical cleavage in the absence (solid gray) or presence of 5 nM cJun/cJun (blue line).
Figure 4.
Aptamer-19(12–74) binds cJun/cJun with high affinity and specificity, competes with AP-1 DNA for binding cJun, and inhibits cJun/cJun activated transcription in cells.
A) Aptamer-19(12–74) is >100-fold more specific for binding cJun/cJun compared to cJun/cFos. Data from EMSAs were quantified and fit to binding curves; the equilibrium dissociation constant of the aptamer for cJun/cJun is 0.5 nM and for cJun/cFos is >85 nM. B) 2 nM cJun/cJun was incubated with 10 nM 5′-Cy5-labeled AP-1 DNA and either unlabeled AP-1 decoy DNA, mutant AP-1 decoy, AS aptamer-19(12–74), or aptamer-19(12–74) at the concentrations indicated. C) Aptamer-19 prevents cJun/cJun from associating with NFAT bound to an NFAT/AP-1 composite site. dsDNA containing an NFAT/AP-1 composite element can be shifted by NFAT and cJun individually (lanes 2 and 3, respectively) or supershifted as a result of NFAT and cJun cooperativity (lane 4). Either an antisense of aptamer-19(12–74), aptamer-19(12–74), or AP-1 DNA was titrated into the reaction containing NFAT/AP-1 composite DNA, NFAT, and cJun. D) Overexpression of NFATc2 and cJun as well as stimulation with PMA and ionomycin are required for full activation of the IL-2 reporter in cells. Firefly luciferase was normalized to renilla luciferase; bars represent the average of three transfections and error bars represent one standard deviation. E) Aptamer-19(12–74) reduces transcription driven by cJun/cJun and NFATc2 from the IL-2 reporter in cells. Oligonucleotide concentrations were held constant under all conditions by cotransfecting a scrambled oligonucleotide of the same length. Firefly luciferase was normalized by the firefly luciferase plasmid copy number in each pool of transfected cells, as determined by real time PCR. Data were normalized to the IL-2 reporter activity in the presence of AS aptamer-19(12–74). Bars represent the average of three transfections and error bars represent one standard deviation. Asterisks represent statistical significance determined by a paired t-test. (*, p-value<0.032)