Conformational and Thermodynamic Properties of Parallel Intramolecular Triple Helices Containing a DNA, RNA, or 2‘-OMeDNA Third Strand

Asensio, Juan Luis; Carr, Reuben; Brown, Tom; Lane, Andrew N.

doi:10.1021/ja991949s

Cited by 54 publications

(52 citation statements)

References 29 publications

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“…Recent analyses by NMR and FT-IR have shown that D:DD and R:DD triple helices (where DD stands for the DNA double helix, and D: or R: for the DNA or RNA TFO, respectively) have heterogeneous backbone conformations, leading to energetically compromised conformations for certain ribo-and deoxyribonucleotides in the three strands (9 -15). Thermodynamic studies have shown that R:DD triple helices have higher thermal stability than D:DD ones (8,(15)(16)(17)(18), whereas similar free energies and equilibrium constants have been found by isothermal titration calorimetry and EDTA cleavage (19,20). Results in our laboratory and others, with third strand sequences containing a variable number of nucleotides with modified bases or backbones, show thus far nonunderstood differences in affinity as a function of the target sequence and the position of the modified nucleosides in the chain (21)(22)(23).…”

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confidence: 58%

Conformational Diversity Versus Nucleic Acid Triplex Stability, a Combinatorial Study

Bernal-Méndez

Leumann

2001

Journal of Biological Chemistry

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The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines.Triple-stranded DNA and RNA structures were first discovered in the late fifties by Felsenfeld and Rich (1, 2). In 1986, two independent research groups demonstrated that triplex-forming oligonucleotides (TFOs) 1 can be used to specifically recognize a given sequence in a DNA double helix (3, 4), and thus paved the way for their potential use as therapeutics in the antigene strategy and as tools in molecular biology. Polypyrimidine TFOs bind specifically to complementary poly(Pu)⅐poly(Py) double-helix sequences by formation of Hoogsteen base pairs between thymines or protonated cytosines in the TFO and adenines or guanines, respectively, in the poly(Pu) strand of the DNA double helix. The molecular recognition process is highly sensitive to base mismatches, so that a single duplex site can be targeted within megabase DNA (5-7).It has been shown previously that triplexes not only form within the pure DNA or RNA backbone context but also within mixed RNA and DNA strands, although with distinct differences in stability within a given sequence context (8). Recent analyses by NMR and FT-IR have shown that D:DD and R:DD triple helices (where DD stands for the DNA double helix, and D: or R: for the DNA or RNA TFO, respectively) have heterogeneous backbone conformations, leading to energetically compromised conformations for certain ribo-and deoxyribonucleotides in the three strands (9 -15). Thermodynamic studies have shown that R:DD triple helices have higher thermal stability than D:DD ones (8, 15-18), whereas similar...

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supporting

confidence: 58%

Conformational Diversity Versus Nucleic Acid Triplex Stability, a Combinatorial Study

Bernal-Méndez

Leumann

2001

Journal of Biological Chemistry

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“…Structural studies indicate that this modification stabilizes the C3Ј-endo,2Ј-exo configuration of the sugar. This is the optimal conformation for triplex formation by pyrimidine oligonucleotides, and provokes the least perturbation of the underlying duplex (31).…”

mentioning

confidence: 99%

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Puri¹,

Majumdar²,

Cuenoud³

et al. 2001

Journal of Biological Chemistry

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2؉ . Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

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“…First, the stability of TFO against nucleases is a concern, which is universal for all ODN-based technologies. Triplex formation involves conformational changes on TFO sequence and some distortion of the underlying dsDNA (312,313), which leads to the intrinsic instability of triplex structures. Triplex formation with (C, T)-motif TFOs is pH dependent because cytosines must be protonated at the N3 position in slightly acidic media (pH<6) to form two hydrogen bonds with G (314).…”

Section: Rules For Triplex Formationmentioning

confidence: 99%

“…The beneficial effects are probably due to the shift in the conformational equilibrium of the sugar to C3' endo configuration. This conformation imposes minimal distortion on the underlying duplex (313) and thus provide "pre-organization", which has entropic benefits (344). In 2'-O-aminoethyl (2'-AE) modification, both a positive charge and a "pre-organized" sugar conformation can be provided (343).…”

Section: Chemical Modifications On Tfosmentioning

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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Conformational and Thermodynamic Properties of Parallel Intramolecular Triple Helices Containing a DNA, RNA, or 2‘-OMeDNA Third Strand

Cited by 54 publications

References 29 publications

Conformational Diversity Versus Nucleic Acid Triplex Stability, a Combinatorial Study

Conformational Diversity Versus Nucleic Acid Triplex Stability, a Combinatorial Study

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

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