Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides

Cassidy, Rachel A.

doi:10.1093/nar/gkg436

Cited by 9 publications

(6 citation statements)

References 58 publications

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“…These data are consistent with our previous results for the serum stability of a methylphosphonate-derivatized hairpin oligo-2′-O-methylribonucleotide (4) and with oligodeoxyribonucleotides having a 3′-terminal methylphosphonate linkage (15). The combined results show that methylphosphonate linkages located in the 3′-terminal portion of the oligomer can impart considerable protection against degradation by 3′-exonuclease activity found in mammalian sera.…”

Section: T H I S C O N T E N T Isupporting

confidence: 92%

3‘-Methylphosphonate-Modified Oligo-2‘-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture

Prater

Miller

2004

Bioconjugate Chem.

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Antisense oligo-2'-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3'-end of an oligo-2'-O-methylribonucleotide is sufficient to prevent degradation by the 3'-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5'-[(32)P]-labeled methylphosphonate modified oligo-2'-O-methylribonucleotides are taken up by mouse L(929) fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3'-methylphosphonate modified oligo-2'-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5'-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5'-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.

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Section: T H I S C O N T E N T Isupporting

confidence: 92%

3‘-Methylphosphonate-Modified Oligo-2‘-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture

Prater

Miller

2004

Bioconjugate Chem.

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“…While TFO RNA-C2 had a K d that was not significantly different from that of TFO DNA-C2, a modest reduction in K d was observed for TFO RNA-C6, suggesting that the 6-carbon linker further stabilizes binding by allowing better psoralen intercalation, or less likely through direct interactions with the triplex structure, consistent with observations in DNA TFOs [19]. This lack of dramatic enhancement in binding affinity of RNA third strands binding to DNA duplexes has also been observed previously by Han and Dervan [20] and may be due to the RNA TFO's lack of the C-5 methyl group in uridine which likely enhances base stacking and triplex stability [1–3, 18, 21, 22]. …”

Section: Resultssupporting

confidence: 80%

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

Krishnan

2010

Journal of Nucleic Acids

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Triplex-forming oligonucleotides (TFOs) with both DNA and 2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and 2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the human HPRT gene. While TFO binding affinity was not dramatically affected by the type of backbone, psoralen photoreactivity was completely abrogated by the 2′-O-methyl RNA backbone. Photoreactivity was restored when the psoralen was conjugated to the RNA TFO via a 6-carbon linker. In contrast to the B-form DNA of triplexes formed by DNA TFOs, the CD spectra of triplexes formed with 2′-O-methyl RNA TFOs exhibited features of A-form DNA. These results indicate that 2′-O-methyl RNA TFOs induce a partial B-to-A transition in their target DNA sequences which may impair the photoreactivity of a conjugated psoralen and suggest that optimal design of TFOs to target DNA damage may require a balance between binding ability and drug reactivity.

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“…It has been shown that cyanide is an extremely strong ligand for platinum and that adding cyanide to a Pt-G adduct will displace the platinum from guanine (46-50). The t1d-4R , t2d-4Y , and t3d-4Y adducts were isolated from the gel and were treated with 200 mM sodium cyanide.…”

Section: Resultsmentioning

confidence: 99%

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

Campbell

Miller

2009

Bioconjugate Chem.

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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...

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Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides

Cited by 9 publications

References 58 publications

3‘-Methylphosphonate-Modified Oligo-2‘-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture

3‘-Methylphosphonate-Modified Oligo-2‘-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture

Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

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

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