Targeted Generation of DNA Strand Breaks Using Pyrene-Conjugated Triplex-Forming Oligonucleotides

Abstract

Gene targeting by triplex-forming oligonucleotides (TFOs) 1 has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can produce more genotoxic lesions. Here we demonstrate that photoactivated pyrene-conjugated TFOs (pyr-TFOs) induce DNA strand breaks near the pyrene moiety with remarkably high efficiency, and also produce covalent pyrene-DNA adducts. Free radical scavenging experiments demonstrated a role for singlet oxygen activated by the singlet-excited state of pyrene in the mechanism of pyr-TFO-induced DNA damage. In cultured mammalian cells, the effect of photoactivated pyr-TFO-directed DNA damage was to induce mutations, in the form of deletions, ~7-fold over background levels, at the targeted site. Thus, pyr-TFOs represent a potentially powerful new tool for directing DNA strand breaks to specific chromosomal locations for biotechnological and potential clinical applications.Triplex-forming oligonucleotides (TFOs) ‡ have been employed to modulate gene structure and function, both in vitro and in vivo. When conjugated to DNA damaging agents, TFOs can induce site-directed DNA damage resulting in enhanced mutagenesis and recombination [reviewed in (1-8)]. Reactive molecules that have been conjugated to TFOs include, but are not limited to, 2-amino-6-vinylpurine, haloacetyl amide, aryl nitrogen mustard, N 4 ,N 4 -etheno-5-methyldeoxycytidine, and various psoralen derivatives. All of these induce sitespecific cross-linking and/or alkylation in double-stranded DNA (dsDNA) (9-15). Psoralenlinked TFOs (pso-TFOs), which can induce covalent monoadducts on one strand or cross-links with both strands of duplex DNA with high efficiency and sequence specificity, are the most studied of the photoreactive TFOs. Pso-TFOs can yield site-specific DNA damage with high efficiency and have been used to introduce mutations and inactivate target genes. However, the existence of cellular mechanisms for repairing the pso-TFO photoproducts without introduction of sequence errors has limited their utility. † Funding was provided in part from National Institutes of Health (CA93729 to K.M.V.) and National Institute of Environmental Health Sciences (ES007784). 1 Abbreviations: Aprt, adenine phosphoribosyl transferase; DMF, dimethylformamide; DSB, double-strand break; dsDNA, doublestranded DNA; EMSA, electrophoretic mobility shift assay; HMWC, high molecular weight complex; PAH, polycyclic aromatic hydrocarbon; pso-, psoralen; pyr-, pyrene; strand breaks, SBs; TFO, triplex-forming oligonucleotide. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptRecent efforts by several groups have improved the binding affinity and sequence specificity of TFOs in vivo. For example, Puri and colleagues found that 2'-O-aminoethyl (2'-AE) ribose substitutions within p...

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

“…It has to be noted that pyrene is an important fluorophore [55] as well as a photoactive reagent. [56] It is an approved and widely used molecule for DNA and RNA probe construction.…”

Synthesis of C-5, C-2′ and C-4′-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes

“…Sugar/phosphate backbone-and base-modified TFOs [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] but also with the aid of triplex stabilizing ligands, which by covalent incorporation into TFOs may provide a sequenceindependent extra binding motif, have been studied. [23][24][25][26][27][28][29][30] Regarding the ligand discovery, the ability of aminoglycosides in stabilizing the DNA and RNA triplexes and their hybrid triplexes has thoroughly been studied by Arya et al [31][32][33][34] Among this carbohydrate family, neomycin has proven to be the most effective triplex stabilizing groove binder. The stabilization originates from the binding to the Watson-Hoogsteen groove (the groove between the pyrimidine strands), 34 in which the amino groups of neomycin rings II and IV (cf.…”

“…Figure 2) are proposed to be involved in the recognition process. The ligand-induced triplex formation has previously been utilized with covalently conjugated spermine, 27 acridine, 24 benzopyridoindole, 25 benzo[f]quino [3,4-b]quinoxaline (BQQ, the best triplex stabilizing intercalator) 25 and other intercalating molecules, 23,26, [28][29][30] but surprisingly not with neomycin despite its obvious potential. Several synthetic studies of neomycin-conjugated oligonucleotides and their applicability in the duplex formation have, however, been reported.…”

Site-Specific DNA Photocleavage and Photomodulation by Oligonucleotide Conjugates

“…In a recent study (Benfield et al, 2008), TFO-pyrene conjugates were utilized to induce DNA strand breaks near the pyrene moiety. Other than psoralen, this is the only UVactivated ODN-conjugate system that was studied in cells.…”