Supercoiled DNA Minicircles under Double-strand Breaks

Understanding how supercoiled DNA releases intramolecular stress is essential for its functional realization.However,the molecular mechanism underlying the relaxation process remains insufficiently explored.Here we employed MD simulations based on the oxDNA2 model to investigate the relaxation process of a 336-base pair supercoiled minicircular DNA under double-strand breaks with two fixed endpoints.Our simulations show that the conformational changes in the DNA occur continuously,with intramolecular stress release happening abruptly only when the DNA chain traverses the breakage site.The relaxation process is influenced not only by the separation distance between the fixed ends but also their angle.Importantly,we observe an inhibitory effect on the relaxation characterized by small angles,where short terminal loops impede DNA conformational adjustments,preserving the supercoiled structure.These findings elucidate the intricate interplay between DNA conformational change,DNA motion and intramolecular stress release,shedding light on the mechanisms governing the relaxation of supercoiled DNA at the molecular level.

How Small DNA Minicircles Can Be Applied to Construct DNA Nanotubes？

DNA as a life＇s information carrier can be modified into geometrically fine nanostructures via self-assembly of designed nucleotides with specified length. In this work, three DNA minicircles with designed lengths of 48-nt, 50-nt, and 52-nt, are directed to self-assemble into nanotubes after hybridization with staple strands, following the folding strategy with each double crossover （DX） at 2.5 turns. Much smaller DNA minicircles such as the 32-nt ring are highly rigid once they form double helices, therefore they lack the flexibility to form finely ordered nanotubes. In the case of nanotubes comprising of 52-nt minicircles, most nanotubes were 800 nm long and 20% were up to 2 p.m, whereas the nanotubes composed of 50 base pair subunits and 48 base pair subunits with the DX at frustrated 2.5 turns showed relatively shorter nanotubes at 700 and 600 （or 500） nm, respectively.

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.