Triplex Staples:  DNA Double-Strand Cross-Linking at Internal and Terminal Sites Using Psoralen-Containing Triplex-Forming Oligonucleotides

Li, Hong; Broughton-Head, Victoria J.; Peng, Guomei; Powers, Vicki E. C.; Ovens, Matthew J.; Fox, Keith R.; Brown, Tom

doi:10.1021/bc0601875

Cited by 35 publications

(21 citation statements)

References 23 publications

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“…Psoralen can also be attached at either end or at an internal site of a TFO and UV irradiation of these complexes produces interstrand cross-links at adjacent TpA steps. Photoreaction of TFOs containing two psoralens, located at the 5 -and 3 -ends, has been used to create crosslinks at both termini of the TFO (triplex staples), producing complexes that have no free single-stranded ends [28].…”

Section: Increasing Affinitymentioning

confidence: 99%

Formation of stable DNA triplexes

Fox

Brown

2011

Biochemical Society Transactions

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Triple-helical nucleic acids are formed by binding an oligonucleotide within the major groove of duplex DNA. These complexes offer the possibility of designing oligonucleotides which bind to duplex DNA with considerable sequence specificity. However, triple-helix formation with natural nucleotides is limited by (i) the requirement for low pH, (ii) the requirement for homopurine target sequences, and (iii) their relatively low affinity. We have prepared modified oligonucleotides to overcome these limitations, including the addition of positive charges to the sugar and/or base, the inclusion of cytosine analogues, the development of nucleosides for recognition of pyrimidine interruptions and the attachment of one or more cross-linking groups. By these means we are able to generate triplexes which have high affinities at physiological pH at sequences that contain pyrimidine interruptions.

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Section: Increasing Affinitymentioning

confidence: 99%

Formation of stable DNA triplexes

Fox

Brown

2011

Biochemical Society Transactions

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“…Therefore, i t is important to develop a simple, quantitative method to detect triplex formation inside living cells for reliable quantification. Psoralen is a bifunctional photoactive agent that has been used as probes of nucleic acid structure and function, and is minimally harmful to cellular constituents (30). Therefore, conjugation of photoreactive psoralen to a TFO at its 5′ end allows photo-induced cross-linking reaction of the psoralen at the specific sequence where the TFO binds to duplex DNA.…”

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confidence: 99%

Sequence-Specific Triple Helix Formation with Genomic DNA

Guntaka

Mahato

2007

Biochemistry

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We have previously demonstrated site-specific delivery of antiparallel phosphorothioate triplex forming oligonucleotide (TFO) specific to -165 to -141 promoter region of alpha1(I) collagen (abbreviated as APS165) to hepatic stellate cells (HSCs) of fibrotic rats after conjugation with mannose 6-phosphate-bovine serum albumin. However, we still need to determine whether there is correlation between transcription inhibition and triplex formation with genomic DNA. In this study, APS165 was modified with psoralen and the extent of triplex formation with alpha1(I) collagen DNA was determined in naked genomic DNA, isolated nuclei of HSC-T6 cells and whole cells by using a simple real-time PCR based method. In this method, a purification step was added to remove unbound APS165, which eliminated the possible artifacts during real-time PCR. Psoralen photoadduct formation was shown to be essential to retain triplex structure under denaturing conditions. On naked genomic DNA, 82.2% of DNA formed triplex and 36.7% of genomic DNA in isolated nuclei at 90 min contained triplex structure. As quantified by real-time PCR, 50% of genomic DNA in living cells at 12 h postincubation contained triplex structures. Furthermore, the triplex formation was dose-dependent with 26.5% and 50% of DNA having triplex structure at concentrations of 1 microM and 5 microM, respectively. Moreover, on a plasmid pCol-CAT220 containing rat alpha1(I) gene promoter (-225 to +113), 75.3% of triplex formation was observed, which was correlated with a 73.6% of transcription inhibition. These findings will further strengthen the therapeutic applications of APS165.

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“…Psoralen is a bifunctional photoactive agent that has been used as probes of nucleic acid structure and function, and is minimally harmful to cellular constituents (408). Conjugation of psoralen to the 5'end of a TFO allows photo-induced cross-linking reaction of the psoralen at the specific sequence where the TFO binds to duplex DNA.…”

Section: Detection and Quantification Of Triplex Formationmentioning

confidence: 99%

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

Houssein

Mahato

2007

Oligonucleotides

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Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is in urgent need to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remains the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of α1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed.

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Triplex Staples: DNA Double-Strand Cross-Linking at Internal and Terminal Sites Using Psoralen-Containing Triplex-Forming Oligonucleotides

Cited by 35 publications

References 23 publications

Formation of stable DNA triplexes

Formation of stable DNA triplexes

Sequence-Specific Triple Helix Formation with Genomic DNA

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

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