Transplatin-Conjugated Triplex-Forming Oligonucleotides Form Adducts with Both Strands of DNA

Abstract

Triplex-forming oligonucleotides (TFOs) can bind to polypurine•polypyrimidine tracts in DNA and as a consequence, perturb normal functioning of a targeted gene. The effectiveness of such anti-gene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here we report that attachment of N-7-platinated guanine nucleosides to the 3′-and/or 5′-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3′,5′ bis platinated TFOs can result in formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37°C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast high levels (~86%) of adduct formation by platinated 2′-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2′-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinumconjugated TFOs may be good candidates for use as anti-gene agents.Over the past two decades, there has been considerable interest in developing oligonucleotides that can specifically inhibit gene expression. Two approaches that have received much attention are antisense oligonucleotides and siRNA. Both approaches target the RNA product of the expressed gene and ultimately result in destruction of this target. Because the RNA products are continuously produced when the gene is transcribed, the antisense oligonucleotide or siRNA must be present constantly in order to prevent their translation into protein.An alternative approach that directly targets and inhibits gene expression utilizes triplexforming oligonucleotides (TFOs). These oligonucleotides are designed to bind to doublestranded DNA at specific regions within the gene (1-3 ). As a consequence of this binding, transcription of the gene is inhibited or mutations are induced that result in aberrant expression Triplex forming oligonucleotides interact via the major groove by binding to polypurine tracts within the DNA target (1-7 ). Oligopurine TFOs utilize reverse-Hoogsteen hydrogen bonds to form A•A-G and G•G-C triads and are oriented antiparallel to the polypurine tract. Oligopyrimidine TFOs utilize Hoogsteen hydrogen bonds to form T•A-T and C + •G-C triads and are oriented parallel to the polypurine tract. In this case the N-3 of the cytosine of the C + •G-C triad must be protonated in order to form a stable Hoogsteen interaction and consequ...

Different Effects of Cisplatin and Transplatin on the Higher-Order Structure of DNA and Gene Expression

“…Accordingly, a partially folded DNA string might allow the monofunctional adducts to form bifunctional adducts of the long-range crosslinks, which can be rearranged to form energetically more favorable interstrand crosslinks between complementary GC base pairs [24]. This hypothesis is supported by the fact that transplatin is more reactive than cisplatin; that a relatively large proportion (~50%) of all adducts on the double-stranded DNA are monofunctional [23]; and that transplatin, owing to its geometry, is a useful tool to prepare a DNA triplex with interstrand GG or GC crosslinking [68,69]. genome-sized DNA is shown in Figure 10.…”

Interaction of metal complexes with nucleic acids

“…29 In addition to the above ''free'' platinating agents, a covalent nucleobase modification of a ss-DNA with a NH 2 -terminating linker coordinated to a Pt II (NH 3 ) 2 Cl unit yielded a monofunctional agent that could coordinate to a G-N7 from a DNA duplex, resulting in triplex assembly. 30,31 The exact stereoelectronic parameters of each complex affect its ability to interact with specific loci or in different modes with DNA prior to inducing platination. As such, computational methods are proving useful in the design of new molecules or prediction of their binding behaviour.…”

Preparation and Application of Triple Helix Forming Oligonucleotides and Single Strand Oligonucleotide Donors for Gene Correction

“…The recognition of a “triplex” binding code (T·A:T, C + ·G:C for pyrimidine motif triplexes; A·A:T, G·G:C for purine motif triplexes), and the development of procedures and instruments for facile oligonucleotide synthesis, was the basis for the concept of triplex forming oligonucleotides (TFOs) as gene targeting reagents [2, 3]. These could introduce damage into specific sequences in the genome of living cells, either as carriers of DNA reactive moieties [4–7] or because the triplex structure might be recognized by cellular activities that would introduce nicks and breaks in an effort to “repair” the triplex [8]. Both scenarios could yield mutations at the site or, if accompanied by an informational donor that could enter an appropriate repair pathway, direct a change in the genomic sequence.…”