Destabilizing Universal Linkers for Signal Amplification in Self-Ligating Probes for RNA

Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4−5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays

Dumbbell-Shaped Nanocircular RNAs for RNA Interference

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops. RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA

Synthesis of the <i>C</i>-Glycosidic Analogue of Adenophostin A and Its Uracil Congener as Potential IP₃ Receptor Ligands. Stereoselective Construction of the <i>C</i>-Glycosidic Structure by a Temporary Silicon-Tethered Radical Coupling Reaction^†

Synthesis of the C-glycosidic analogue 9 of adenophostin A, a very potent IP3 receptor agonist, and its uracil congener 10 was achieved via a temporary silicon-tethered radical coupling reaction as the key step. Phenyl 3,4,6-tri-O-(p-methoxybenzyl)-1-seleno-β-d-glucopyranoside (27) and 3-deoxy-3-methylene-1,2-O-isopropylidene-α-d-erythro-pentofuranose (30) were connected by a dimethylsilyl tether to give the radical coupling reaction substrate 24, which was successively treated with Bu3SnH/AIBN in benzene and TBAF in THF to give the coupling product 25 with the desired (3α,1‘α)-configuration as the major product. From 25, the targets 9 and 10 were synthesized via introduction of adenine or uracil base by Vorbrüggen's method and phosphorylation of the hydroxyls by the phosphoramidite method

Long-Lived Luminogenic Probe for Detection of RNA in a Crude Solution of Living Bacterial Cells

A pre-type sensitizer for a lanthanide complex on an oligonucleotide was successfully converted to a perfect final structure in a target DNA/RNA-templated reaction, without any chemical reagent or enzyme, under neutral conditions. The final form of the lanthanide–oligonucleotide provided a long-lived luminescence signal, appropriate for time-gated luminescence analysis and signal amplification. Target DNA/RNA-assisted time-gated luminescence analysis is a powerful tool for elimination of autofluorescence and detection of target RNA in living bacterial cells

Highly α- and β-Selective Radical<i> C</i>-Glycosylation Reactions Using a Controlling Anomeric Effect Based on the Conformational Restriction Strategy. A Study on the Conformation−Anomeric Effect− Stereoselectivity Relationship in Anomeric Radical Reactions

We hypothesized that, because the stereoselectivity of anomeric radical reactions was significantly influenced by the anomeric effect, which can be controlled by restricting the conformation of the radical intermediate, the proper conformational restriction of the pyranose ring of the substrates would therefore make highly α- and β-stereoselective anomeric radical reactions possible. Thus, the conformationally restricted 1-phenylseleno-d-xylose derivatives 9 and 10, restricted in a 4C1-conformation, and 11 and 12, restricted in a 1C4-conformation, were designed and synthesized by introducing the proper protecting groups on the hydroxyl groups on the pyranose ring as model substrates for the anomeric radical reactions. The radical deuterations with Bu3SnD and the C-glycosylation with Bu3SnCH2CHCH2 or CH2CHCN, using the 4C1-restricted substrates 9 and 10, afforded the corresponding α-products (α/β = 97:3−85:15) highly stereoselectively, whereas the 1C4-restricted substrates 11 and 12 selectively gave the β-products (α/β = 1:99−0:100). Thus, stereoselectivity was significantly increased by conformational restriction and was completely inverted by changing the substrate conformation from the 4C1-form into the 1C4-form. Ab initio calculations suggested that the radical intermediates produced from these substrates possessed the typical 4C1- or 1C4-conformation, which was similar to that of the substrates, and that the anomeric effect in these conformations would be the factor controlling the transition state of the reaction. Therefore, the highly α- and β-selective reactions would occur because of the anomeric effect, which could be manipulated by conformational restriction of the substrates, as expected. This would be the first radical C-glycosylation reaction to provide both α- and β-C-glycosides highly stereoselectively

Factor decay rate related to improved self-efficacy among psychiatric nurses.

Factor decay rate related to improved self-efficacy among psychiatric nurses.</p

Highly Stereoselective Grignard Addition to <i>Cis</i>-Substituted <i>C</i>-Cyclopropylaldonitrones. The Bisected <i>s</i>-<i>Trans</i> Transition State Can Be Stabilized Effectively by the Lewis Acid-Coordination

We previously found that Grignard addition to a C-cyclopropylaldonitrone, C-[cis-2-(N,N-diethylcarbamoyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (1), stereoselectively gave the anti-product 3, in which the stereoselectivity was particularly high when MgBr2 was the additive. In this study, the reaction pathway was investigated in detail. The stereoselective addition was initially thought to occur via either a 1,5-chelation-controlled or a bisected s-trans conformation-controlled pathway. However, Grignard addition to a nonchelating silyl ether-type substrate, C-[cis-2-(tert-butyldiphenylsilyloxymethyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (7), also gave the anti-product 9 with high stereoselectivity suggesting that chelation is not important in the reaction. Theoretical calculations of C-cyclopropylaldonitrones showed that the coordination of Mg2+ at the nitrone oxygen significantly stabilizes the bisected s-trans conformer due to the effective hyperconjugation between the π* of the nitrone CN bond and the electron-donating cyclopropane orbitals. This kind of orbital interaction is able to stabilize the transition state of the nucleophilic addition and is maximized in the bisected conformation, in which the orbitals of the forming bond and the cyclopropane C−C bond are in an almost planar arrangement. Thus, the high stereoselectivity can be explained by nucleophilic attack on the less hindered side of the CN bond of the substrates in the Mg2+−coordinated bisected s-trans conformation

Factors related to decreased self-efficacy among psychiatric nurses (n = 132).

Factors related to decreased self-efficacy among psychiatric nurses (n = 132).</p

The factors related to self-efficacy and the GSES-J correlations.

The factors related to self-efficacy and the GSES-J correlations.</p

Mechanistic Study of the Ring-Enlargement Reaction of (3-Oxa-2-silacyclopentyl)methyl Radicals into 4-Oxa-3-silacyclohexyl Radicals. Evidence for a Pentavalent Silicon-Bridging Radical Transition State in 1,2-Rearrangement Reactions of β-Silyl Radicals

A mechanistic study was performed on a novel radical ring-enlargement reaction of (3-oxa-2-silacyclopentyl)methyl radicals into 4-oxa-3-silacyclohexyl radicals. Two pathways, one via a pentavalent silicon-bridging radical transition state (or intermediate), the other via β-elimination to give a ring-opened silyl radical, can be postulated. The radical reactions of 1 and 2, which are precursors for a (3-oxa-2-silacyclopentyl)methyl radical C‘ and a 4-oxa-3-silacyclohexyl radical D‘, respectively, showed that the ring-enlargement rearrangement of C‘ into D‘ is irreversible. 1H NMR analysis of the radical reactions of 8a and 8b, which have an asymmetric center at silicon, indicated that the configuration at the silicon atom is retained via a pentavalent silicon-bridging radical transition state (or intermediate) during the ring-enlargement reaction. Furthermore, examination of the radical ring-enlargement reaction with a deuterium-labeled substrate 12D showed that the ring-enlargement reaction did not involve β-elimination to give a ring-opened silyl radical. Based on these results, we conclude that the ring-enlargement reaction occurs via a pentavalent silicon-bridging radical transition state (or intermediate). This is the first experimental evidence for such a pentavalent silicon radical, which has been previously postulated to understand radical reactions of organic silicon compounds