Rula Zain

Nucleoside H-phosphonates. X: Studies on nucleoside hydrogenphosphonothioate diester synthesis

Abstract Synthesis and chemical properties of nucleoside H-phosphonothioates are discussed in the context of possible application of these compounds as intermediates in the synthesis of oligonucleotide analogues.

Do natural DNA triple-helical structures occur and function in vivo?

Abstract: Formation of natural intramolecular triple-helical structures of DNA is still an intriguing research topic in view of the possible involvement of these structures in biological processes. The biochemical and biophysical properties of DNA triplex structures have been extensively studied, and experimental data show that H-DNA is likely to form in vivo and may regulate the expression of various genes. However, direct and unambiguous evidence of the possible biological roles of these structures is yet elusive. This review focuses on the basic facts that are in favor of, or against, the hypothesis of the presence and function of natural DNA triple-helical structures in vivo, and outlines the different methods and probes that have been used to support these facts.

Design of a triple-helix-specific cleaving reagent.

BACKGROUND Double-helical DNA can be recognized sequence specifically by oligonucleotides that bind in the major groove, forming a local triple helix. Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive study. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We previously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) can strongly stabilize triple helices. RESULTS We have designed and synthesized the first triple-helix-specific DNA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacetic acid (EDTA). The intercalative binding of BQQ should position EDTA in the minor groove of the triple helix. In the presence of Fe(2+) and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) DNA fragment at the site where an oligonucleotide binds to form a local triple helix. The selectivity of the BQQ-EDTA conjugate for a triplex structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA. CONCLUSIONS This new class of structure-directed DNA cleaving reagents could be useful for cleaving DNA at specific sequences in the presence of a site-specific, triple-helix-forming oligonucleotide and also for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.

Nucleoside Phosphonates. Development of Synthetic Methods and Reagents

Abstract In this paper a short account of our recent research concerning development of new synthetic methods and new reagents for the preparation of DNA and RNA fragments and their analogues is given. #Dedicated to Professor Yoshihisa Mizuno on the occasion of his 75th birthday. ¶Present address: Astra Production Chemicals, S-151 85 Sodertalje, Sweden.

Stereospecific oxidation and oxidative coupling of H-phosphonate and H-phosphonothioate diesters

Abstract Oxidation of dinucleoside H-phosphonothioate diesters with the aid of iodine in aqueous acetonitrile and triethylamine, and oxidative coupling of H-phosphonate and H-phosphonothioate diesters with ethanol under similar conditions in anhydrous acetonitrile were found to be stereospecific reactions.

A new entry to nucleoside phosphorofluoridate and nucleoside phosphorofluoridothioate diesters

Abstract Oxidation of H-phosphonate or H-phosphonothioate diesters with iodine in the presence of triethylamine trishydrofluoride furnished a rapid and quantitative formation of the corresponding phosphorofluoridate or phosphorofluoridothioate diesters.

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.

Triple-helix directed cleavage of double-stranded DNA by benzoquinoquinoxaline-1,10-phenanthroline conjugates.

Oligonucleotide‐directed triple‐helix formation provides a rational means to interfere with genomic DNA targets and to direct modifications at specific sites. We have developed a new class of compounds that, at low concentrations, efficiently targets and damages double‐stranded DNA specifically at the site where a triple‐helical structure is formed. In these new compounds, a triple‐helix‐specific intercalator—benzoquinoquinoxaline (BQQ)—was coupled to one of two isomeric 1,10‐phenanthrolinecarboxaldehyde derivatives. 1,10‐Phenanthroline derivatives are known to cleave DNA in the presence of copper ions. The obtained BQQ‐1,10‐phenanthroline (BQQ–OP) conjugates were compared with regard to their ability to cleave triple‐helix DNA. Both conjugates displayed a sequence preference inside the triple‐helical site, as judged from the more pronounced cleavage obtained at stretches of T⋅A×T base triplets.

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Background: DNA triplex helix structures are alternate DNA structures that can be a source of genomic instability. Results: ChlR1 helicase has a novel and distinct triplex DNA unwinding activity. Conclusion: ChlR1 defends genome integrity by resolving triplex DNA structures. Significance: The processing of triplex DNA substrates by proteins such as ChlR1 plays critical roles in genome maintenance. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.

Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreichs ataxia. (GAA)n repeats form non‐B‐DNA structures, including triple helix H‐DNA and higher‐order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non‐B‐DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)n binding by triplex‐stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex‐DNA‐cleaving BQQ‐1,10‐phenanthroline (BQQ‐OP) compounds. We also examined the ability of these compounds to act as structural probes for H‐DNA formation within higher‐order structures at pathological frataxin sequences in plasmids. DNA‐complex‐formation analyses with a gel‐mobility‐shift assay and sequence‐specific probing of H‐DNA‐forming (GAA)n sequences by single‐strand oligonucleotides and triplex‐directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)n repeats under physiologically relevant conditions.