Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems

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

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

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

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

Chemical formula of the DNA cleavage reaction.

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