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Supplementary METHODS
Crosslink formation in the supF reporter gene. The pSupFG1 plasmid was incubated with
either the specific TFO (pAG30) or a control TFO (pSCR30) in triplex binding buffer for 4
hour at 37ºC. Samples were irradiated with UVA light (365 nm, 1.8 J/cm2) to induce
psoralen ICLs at the target site in the plasmid DNA. A 188 bp plasmid fragment surrounding
the site-specific ICL was released by digestion with EcoRI and SacI enzymes, radiolabeled at
the 5’ ends using T4 polynucleotide kinase and [-32P]-ATP, and subjected to 8% denaturing
PAGE to separate the crosslinked substrate from unadducted substrate DNA. Radioactivity
was quantified using a PhosphorImager.
Repair efficiency in the vicinity of TFO-targeted psoralen ICL. Plasmid, pSupFG1,
containing psoralen ICLs, UVC-induced damage, or no damage was subjected to an in vitro
repair assay as described in the Methods section. Then the plasmids were digested with
EcoRI and SacI to release the 188 bp fragment surrounding the site-specific ICL.
Visualization of the plasmid DNA and the incorporated [-32P]-dCTP was achieved by
ethidium bromide staining and autoradiography, respectively, and quantification was
performed using a PhosphorImager. The amount of radioactivity incorporated in the vicinity
of the psoralen ICL was calculated by dividing the amount of radioactivity in the 188 bp
fragment from the damaged pSupGF1 plasmid by the amount of damaged pSupFG1 DNA
loaded. To calculate the fold induction in repair, the amount of radioactivity (normalized to
the amount of DNA loaded) obtained from the undamaged pSupFG1 plasmid was set to one.
Supplementary Figure Legends
Supplementary Figure 1. Efficiency of targeted psoralen ICL formation in the supF gene.
Lane 1 contains the pSupFG1 plasmid in the absence of TFO treatment; lane 2 contains
pSupFG1 plasmid incubated with the specific psoralen-modified TFO, pAG30; lane 3
contains pSupFG1 plasmid incubated with the control psoralen-modified TFO, pSCR30. All
samples were exposed to UVA (1.8 J/cm2). A fragment containing the psoralen-crosslinking
site was digested from the plasmids, radiolabeled, and subjected to denaturing PAGE.
Supplementary Figure 2. Repair efficiency in the vicinity of the psoralen ICL in the
supF gene in MMR-deficient and MMR-proficient human cell extracts. (A)
autoradiogram (left panel) and ethidium bromide stained gel (right panel) demonstrating
DNA repair efficiencies measured by incorporation of radiolabeled nucleotides into the
188 digested fragment of pSupFG1 plasmid. (B) Fold induction of radioactivity
incorporated into the digested fragment of the damaged plasmid over that incorporated in
the undamaged pSupFG1 plasmid.