Assembling nanostructures from DNA using a composite nanotweezers with a shape memory effect

The article demonstrates a technique for fabricating a structure with the inclusion of suspended DNA threads and manipulating them using composite nanotweezers with shape memory effect. This technique could be suitable for stretching of nanothin DNA-like conductive threads and for measuring their electrical conductivity, including the I-V characteristic directly in the electron microscope chamber, where the nanotweezers provide a two-sided clamping of the DNA tip, giving a stable nanocontact to the DNA bundle. Such contact, as a part of 1D nanostructure, is more reliable during manipulations with nanothreads than traditional measurements when a nanothread is touched by a thin needle, for example, in a scanning tunnel microscope.Comment: To be presented on IEEE 3M-NANO 201

SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish Embryos

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence

A Comparison of Genome-Wide DNA Methylation Patterns between Different Vascular Tissues from Patients with Coronary Heart Disease

<div><p>Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the <i>MIR10B</i> gene sequence and about 1 kb upstream of the <i>HOXD4</i> gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.</p></div

Flow chart describing experiment and analysis.

<p>Flow chart describing experiment and analysis.</p

Methylation level (mean ±SD) at the promoter of <i>HOXD4</i> gene in paired vascular tissues from twenty one patients with atherosclerosis.

<p>CAP indicates right coronary arteries in the area of advanced atherosclerotic plaques; IMA, internal mammary arteries; GSV, great saphenous veins.</p><p>Methylation level (mean ±SD) at the promoter of <i>HOXD4</i> gene in paired vascular tissues from twenty one patients with atherosclerosis.</p

Volcano plot of - log10 (P-value) against delta beta value, representing the methylation difference between great saphenous veins (GSV) and internal mammary arteries (IMA).

<p>A total of 200 CpG-sites hypermethylated in GSV with a delta beta ≥ 0.2 and FDR-adjusted p<0.05 are shown in red. A total of 135 CpG-sites hypomethylated in GSV with a delta beta ≤ -0.2 and FDR-adjusted p<0.05 are shown in blue. CpG-sites that exhibited a methylation level difference less 20% are shown in grey. Light red and light blue colors indicate highly differentially methylated CpG-sites (delta beta ≥ 0.40 or delta beta ≤ -0.40 with FDR-adjusted p<0.05). Dashed lines indicate cut-offs for significance.</p

Heatmap analysis of 22 highly differentially methylated CpG-sites (delta beta ≥ 0.40 or delta beta ≤ -0.40 with FDR-adjusted p<0.05) between right coronary arteries in the area of advanced atherosclerotic plaques (CAP) and internal mammary arteries (IMA).

<p>Regions shaded blue in the heat map represent hypomethylated regions, regions shaded red represent hypermethylated regions. The top black rectangles shows columns representing CpG-sites located inside CpG-island. Gene symbols and CpG-site IDs are shown on the bottom. Sample IDs are on the right.</p

The mean chronological age and DNA methylation age (DNAm) of vascular tissues: right coronary artery in the area of advanced atherosclerotic plaques (CAP), internal mammary arteries (IMA), and great saphenous veins (GSV).

<p>The mean chronological age and DNA methylation age (DNAm) of vascular tissues: right coronary artery in the area of advanced atherosclerotic plaques (CAP), internal mammary arteries (IMA), and great saphenous veins (GSV).</p