Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells

Degols, Geneviève; Clarenc, J P; Lebleu, Bernard; Leonetti, Jean Paul

doi:10.1016/s0021-9258(19)89479-6

Cited by 47 publications

(9 citation statements)

References 23 publications

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“…Previous reports (29,30,32,47,48) indicate that free psoralen preferentially cross-links 5′-TpA and 5′-ApT sites, with 5′-TpA sites favored by at least 10-fold. Most studies using psoralen-conjugated TFOs have targeted 5′-TpA sites located at the duplex-triplex junction or immediately adjacent to it and have demonstrated cross-linking efficiencies approaching 100% (50,(53)(54)(55), but more typically in the range of 25-80% (46,(56)(57)(58)(59). In this study, two 5′-ApT sites at the junction were cross-linked with efficiencies of 56 and 65% (Figure 3).…”

Section: Discussionmentioning

confidence: 57%

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

1999

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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.

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Section: Discussionmentioning

confidence: 57%

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

1999

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“…Thus, for example, attachment of a DNA duplex-intercalating chromophore to the TFO can effectively “clamp” the third strand to the target by binding to duplex regions near the triplex−duplex junction ,5c,6d, …”

Section: Introductionmentioning

confidence: 99%

Molecular Anchoring of Duplex and Triplex DNA by Disubstituted Anthracene-9,10-diones: Calorimetric, UV Melting, and Competition Dialysis Studies

et al. 1996

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Isothermal titration calorimetry, UV melting, and competition dialysis techniques have been used to examine the binding of isomeric 1,4- and 2,6-bis(ω-aminopropionamido)-substituted anthracene-9,10-diones (anthraquinones) with dA n ·dT n duplexes and dT n −dA n ·dT n triplexes. Recent footprinting studies [Fox, K. R.; Polucci, P.; Jenkins, T. C.; Neidle, S. Proc. Natl. Acad. Sci. U.S.A. 1995 , 92, 7887−7891] indicate that 2,6 derivatives, but not their 1,4 counterparts, differentially stabilize triple-stranded DNA and may have application in antigene chemotherapy. Thermodynamic investigations are here reported for interaction with dA18·dT18 and dT18−dA18·dT18. The 2,6 compound shows preferential triplex binding, with K b values of 1.8 × 104 M (duplex)-1 and 2.2 × 105 M (triplex)-1 at 25 °C in aqueous solution, pH 6.0, whereas the 1,4 isomer favors duplex binding, with K b values of 1.1 × 105 M (duplex)-1 and 3.5 × 104 M (triplex)-1. Binding to the preferred DNA is enthalpically driven for each ligand, whereas binding to the disfavored DNA is either entropically driven or enthalpy/entropy compensated. Further, the binding site sizes (3.6 base pairs/base triplets) suggest DNA intercalation. Competition dialysis studies with poly(dA)·poly(dT) and poly(dA)·poly(dT)2 confirm these binding preferences, and qualitative support is provided from UV melting experiments. Such studies reveal triplex disruption by the 1,4 isomer at low drug concentrations while the 2,6 compound effects stabilization toward thermal triplex denaturation. Spectrophotometric studies of each free ligand indicate self-association in aqueous solution, with dimerization constants at 25 °C of (2.9 ± 0.2) × 103 and (3.2 ± 0.1) × 103 M-1 respectively for the 1,4 and 2,6 isomers. Taken together, these data provide a firm thermodynamic basis for the contrasting duplex/triplex binding preferences of this isomeric family of ligands.

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“…Oligodeoxyribonucleotides can interact with purine rich sequences in double-stranded DNA to form triple-stranded complexes (for recent reviews see [1][2][3][4]. These interactions may be useful in designing 'antigene' oligonucleotides which can specifically bind to DNA for the purposes of modifying or controlling gene expression (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Two types of triplex 'motifs' are currently recognized, the pyr•pur•pyr motif and the pur•pur•pyr motif.…”

Section: Introductionmentioning

confidence: 99%

Triplex Formation by a Psoralen-Conjugated Oligodeoxyribonucleotide Containing the Base Analog 8-Oxo-Adenine

Miller¹,

Bi²,

Kipp³

et al. 1996

Nucleic Acids Research

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Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.

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Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells

Cited by 47 publications

References 23 publications

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

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

Molecular Anchoring of Duplex and Triplex DNA by Disubstituted Anthracene-9,10-diones: Calorimetric, UV Melting, and Competition Dialysis Studies

Triplex Formation by a Psoralen-Conjugated Oligodeoxyribonucleotide Containing the Base Analog 8-Oxo-Adenine

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