Psoralen Photo-Cross-Linking by Triplex-Forming Oligonucleotides at Multiple Sites in the Human Rhodopsin Gene

Abstract

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.

Activation of gene expression by triplex-directed psoralen crosslinks

“…It includes a total of 14 carbons plus a disulfide bond, instead of the 6 carbons in pso-TFO2. Long linkers can lower crosslinking efficiency (Perkins et al, 1999;Raha et al, 1998). No crosslinks were observed when the DNA was irradiated in the absence of a psoralen-conjugated TFO (data not shown).…”

“…Psoralen-conjugated TFO2 (pso-TFO2) formed photoadducts efficiently (>75%) with its duplex target in previous studies, and most of the photoadducts were interstrand crosslinks (Perkins et al, 1999). The adapter Rh2A is 52 nucleotides long and contains a TFO2 domain connected via 3 Ts to a sequence that can form a double-stranded hairpin containing one E/GRE enhancer.…”

Site-Specific DNA Photocleavage and Photomodulation by Oligonucleotide Conjugates

“…Concerning the psoralen derivative attached to the TFO, besides the chemical modification of psoralen structure, a 5 0 -TpA-3 0 or 5 0 -ApT-3 0 sequence should be present adjacent to triplex-duplex junction with psoralen bound at the 5 0 end of the TFO (Takasugi et al, 1991;Perkins et al, 1999). A proposed reaction mechanism suggests a monoadduct formation with one DNA strand, followed by interstrand crosslink (Oh and Hanawalt, 2000).…”

“…The rate of photochemically induced monoadduct formation (k 1 ) increases with improved triplex stability (Takasugi et al, 1991;Giovannangeli et al, 1992;Perkins et al, 1999) and is therefore strongly dependent on TFO concentration, whereas k 2 is independent on TFO concentration and may be influenced by the external macromolecular environment (Oh et al, 2008).…”

“…These results emphasize the importance of covalent adduct formation with the template strand for efficient suppression of transcriptional elongation. Blocking gene expression induced by TFO-pso was examined in the case of the rhodopsin gene (Perkins et al, 1999). Mutations within the rhodopsin gene result in expression of mutant rhodopsin, which is believed to be responsible for autosomal dominant retinitis pigmentosa, a disease that results in blindness.…”