Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivit

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivit

Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells

A DNA tetrahedron is employed for efficient delivery of doxorubicin into drug-resistant breast cancer cells. The drug delivered with the DNA nanoconstruct is considerably cytotoxic, whereas free doxorubicin is virtually non-cytotoxic for the drug-resistant cells. Thus, the DNA tetrahedron, made of the inherently natural and biocompatible material, can be a good candidate for the drug carrier to overcome MDR in cancer cells.close11

Superior patient survival for continuous ambulatory peritoneal dialysis patients treated with a peritoneal dialysis fluid with neutral pH and low glucose degradation product concentration (Balance)

BACKGROUND: In recent years, laboratory and clinical research has suggested the need for peritoneal dialysis fluids (PDFs) that are more biocompatible than the conventional PDFs commonly used today. Bioincompatibility of PDF has been attributed to low pH, lactate, glucose, glucose degradation products (GDPs), and osmolality. PDFs with neutral pH and low GDPs are now available commercially. In vitro and early clinical studies suggest that these solutions are indeed more biocompatible but, as of now, there is no evidence that their use improves patient outcome. METHODS: Using a dedicated database of over 2000 patients treated with PD in Korea, we were able to conduct a retrospective observational study comparing outcomes for incident continuous ambulatory PD patients treated with a standard, conventional, heat-sterilized PDF to the outcomes for patients treated with a novel, low GDP, neutral-pH PDF prepared in a dual-compartment, double-bag PD system (Balance; Fresenius Medical Care, St. Wendel, Germany). In an intention-to-treat analysis, patient and technique survival, peritonitis-free survival, and peritonitis rates were compared in 611 patients treated with Balance for up to 30 months and 551 patients with a standard PDF (stay . safe; Fresenius Medical Care) treated in the same era and with equivalent follow-up. RESULTS: The patients were well matched for most relevant characteristics except older age distribution for the patients treated with the standard PDF. Patients treated with Balance had significantly superior survival compared to those treated with the standard PDF (74% vs 62% at 28 months, p = 0.0032). In a multivariate Cox regression model including age, diabetes, and gender, the survival advantage persisted (relative risk of death for Balance 0.75, 95% confidence interval 0.56 - 0.99, p = 0.0465). Modality technique survival was similarin Kaplan-Meieranalysis for both PDFs. No differences were detected in peritonitis-free survival or in peritonitis rates between the two solutions. CONCLUSION: This study, for the first time, suggests that treatment with a novel biocompatible PDF with low GDP concentration and neutral pH confers a significant survival advantage. The exact mechanisms for such a survival advantage cannot be determined from this study. The usual criticisms of observational studies apply and the results reported here strongly warrant the undertaking of appropriately designed, randomized, controlled clinical trials

Modulation of p300 binding by posttranslational modifications of the C-terminal activation domain of hypoxia-inducible factor-1α

AbstractPosttranslational modifications of hypoxia-inducible factor-1α (HIF-1α) influence HIF-mediated transcription, likely by affecting binding to p300/cAMP-response element-binding protein (CBP). To systematically analyze the HIF-1α–p300/CBP interaction, we developed a fluorescence polarization-based binding assay, employing fluorescein-labeled peptides derived from the C-terminal transactivation domain (C-TAD) of HIF-1α. After optimized for effectively capturing p300/CBP, the assay was utilized for evaluating direct effects of posttranslational modifications of the HIF-1α C-TAD on p300 binding. The results demonstrated that asparagine hydroxylation and S-nitrosylation of HIF-1α decrease p300 binding, while its phosphorylation does not affect p300 binding, which was reconfirmed by competitive inhibition analyses using mutant peptides

Bicyclo[3.2.1]amide-DNA: a chiral, nonchiroselective base-pairing system

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analog, are reported. This analog consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleobases were attached through an extended, acyclic amide linker. Homobasic adenine-contg. bca decamers form duplexes with complementary oligonucleotides contg. bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed mol. modeling study of left- and right-handed bca-DNA-contg. duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA assocn. [on SciFinder (R)