Triazolyl Donor/Acceptor Chromophore Decorated Unnatural Nucleosides and Oligonucleotides with Duplex Stability Comparable to That of a Natural Adenine/Thymine Pair

We report the design and synthesis of triazolyl donor/acceptor unnatural nucleosides via click chemistry and studies on the duplex stabilization of DNA containing two such new nucleosides. The observed duplex stabilization among the self-pair/heteropair has been found to be comparable to that of a natural A/T pair. Our observations on the comparable duplex stabilization has been explained on the basis of possible π–π stacking and/or charge transfer interactions between the pairing partners. The evidence of ground-state charge transfer complexation came from the UV–vis spectra and the static quenching of fluorescence in a heteropair. We have also exploited one of our unnatural DNAs in stabilizing abasic DNA

Triazolyl Donor/Acceptor Chromophore Decorated Unnatural Nucleosides and Oligonucleotides with Duplex Stability Comparable to That of a Natural Adenine/Thymine Pair

We report the design and synthesis of triazolyl donor/acceptor unnatural nucleosides via click chemistry and studies on the duplex stabilization of DNA containing two such new nucleosides. The observed duplex stabilization among the self-pair/heteropair has been found to be comparable to that of a natural A/T pair. Our observations on the comparable duplex stabilization has been explained on the basis of possible π–π stacking and/or charge transfer interactions between the pairing partners. The evidence of ground-state charge transfer complexation came from the UV–vis spectra and the static quenching of fluorescence in a heteropair. We have also exploited one of our unnatural DNAs in stabilizing abasic DNA

Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div

Chemical formula of the DNA cleavage reaction.

<p>R is expected to be an abasic sugar derivative.</p

Chemical structures of thymine (T) and 5-ethynyluracil (EU).

<p>Chemical structures of thymine (T) and 5-ethynyluracil (EU).</p

Degradation of DNA oligonucleotides containing 5-ethynyluracil.

<p>(A), (B) HPLC charts of T₆(EU)T₆ before (gray) and after (black) the reaction in 14% NH₃aq (A) or 20% MeNH₂aq (B) at 70°C for 2 hours. (C), (D) (EU)T₂AT₂GT₂ (C) and T₂AT₂GT₂(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH₂aq at 70°C for 2 hours.</p

Construction of plasmid from two PCR-amplified DNA fragments.

<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH₂ was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p

Multisite phosphorylation of CaMKIIβ underlies multistep sleep regulation.

(A) Sleep/wake parameters of mice expressing the quadruple-phosphomimetic CaMKIIβ mutants related to T311. Multiple comparison tests were performed between all individual groups. Error bars: SEM, *p p p (B) Expression levels of each mutant in the 293T analyzed by the dot blotting of cell lysates. The values are shown in the mean ± SD (n = 2, independent experiments). (C) In vitro kinase activity of CaMKIIβ phosphomimetic mutants. Phosphorylation (%) indicates the percentage of the phosphorylated substrate relative to the total peptide in the presence or absence of CaM. The represented values are the mean ± SD (n = 2, independent experiments). The relative amount of CaMKIIβ in the cell lysates was adjusted to be the same level as WT. The mutants with blue labels exhibited B). The mutants with green labels exhibited (D) Time series changes of sleep-controlling residues phosphorylation under different Ca2+ conditions in vitro. The represented values are the mean ± SD (n = 2 independent experiments). The signal intensity of the detected peptides was normalized to the maximum value in the time series. The quantified values at 0 min were obtained from the sample before adding CaM and were shared in every Ca2+ conditions. The dashed lines trace the dynamics of T287 phosphorylation in the 0.5 mM Ca2+ condition. (E) Example chromatogram of SRM measurement for pS182 peptide. The red line indicates the retention time of a peptide phosphorylated at S182. The blue line indicates the retention time of a peptide phosphorylated at T177. (F) S26-, T306-, T307-, or T311-phosphorylated peptides from 293T cell lysates. The 293T cells were transfected with plasmids overexpressing CaMKIIβ WT, T287D, or K43R:T287D. Each peptide was quantified by SRM methods and normalized to the level of total CaMKIIβ, which was also quantified by SRM methods for analyzing the CaMKIIβ-derived unphosphorylated peptides. The values are shown as mean ± SD. The underlying data can be found in S1 Data. Quantified values of CaMKIIβ peptides for S12D Fig are provided in S4 Data file. See also S3 Table. CaM, calmodulin; CaMKIIβ, calmodulin-dependent protein kinase IIβ; SRM, selected reaction monitoring; WT, wild-type. (PDF)</p

Sleep induction by CaMKIIβ T287D mutant is independent from core circadian clock genes.

(A-D) Sleep/wake parameters and sleep profiles, averaged over 4 days, of Cry1/2 DKO mice expressing WT CaMKIIβ or the T287D mutant under the LD condition (A and B) or DD (C and D). Multiple comparison tests were performed between all individual groups. (E-H) Sleep/wake parameters and sleep profiles, averaged over 4 days, of Per1/2 DKO mice expressing WT CaMKIIβ or the T287D mutant under the LD condition (E and F) or DD (G and H). Multiple comparison tests were performed between all individual groups. The underlying data can be found in S1 Data. Error bars: SEM, *p p p < 0.001, n.s.: no significance. CaMKIIβ, calmodulin-dependent protein kinase IIβ; CT, circadian time; DD, constant dark; DKO, double knockout; LD, light/dark; WT, wild-type; ZT, zeitgeber time.</p

Expression of the CaMKIIβ throughout the brain by AAV-PHP.eB.

Volume-rendered and single-plane images of the brain expressing H2B-mCherry under hSyn1 promoter by the AAV (mCherry, green) counterstained with RD2 (red). A volume-rendered image is shown in the center. Single-plane and magnified images are shown for cerebral cortex, thalamus, hippocampus, midbrain, cerebellum, striatum, and olfactory bulb. Scale bar in the center image, 3 mm; other scale bars, 100 μm. AAV, adeno-associated virus; CaMKIIβ, calmodulin-dependent protein kinase IIβ; hSyn1, human synapsin-1. (TIFF)</p