Journal: Nature Communications
Article Title: Synthesizing topological structures containing RNA
Figure Lengend Snippet: Constructing ssRNA topological structures with the junction-based method. ( a ) Preparation of the ssRNA strand with uniform ends and proper end groups for the DNA-splinted RNA ligation. ( b ) Both the positive ( TK j ( + )) and negative ( TK j ( − )) RNA trefoil knots of the same sequence are constructed by configuring tensegrity triangles with different handedness. The same scaffolds (blue) are threaded by different staple sets (grey or purple) to form a 17-bp-edged right-handed tensegrity triangle for the positive trefoil knot, or a 14-bp-edged left-handed tensegrity triangle for the negative one, respectively. Each topology is designated by the Alexander–Briggs notation n i or , where n is the minimal number of nodes, C is the number of components (for links), and i distinguishes different topologies with the same n and C . ( c ) dPAGE analysis of TK j ( + ) (lanes 1 and 2), TK j ( − ) (lanes 3 and 4), and their circular ( C j , lanes 5 and 6) and linear ( L j , lanes 7 and 8) counterparts. Lanes 2, 4, 6 and 8 contain samples digested by RNase R. ( d ) The assembly complex for the hybrid BR contains tensegrity triangles of both handedness to generate three positive nodes plus three negative nodes. ( e ) Topological analyses of the hybrid BR. Lane 1, gel-purified BR; lanes 2–6, BR treated by RNase H, Nt.AlwI (for cleaving the red ring), Nt.BspQI (for cleaving the green ring), DNase I, and E. coli DNA Topo I; lanes 7 and 8, DNA and RNA references of the three individual components. During the purification of BR, the breaking down of the 95-nt circular RNA component is unavoidable, and a portion of BR falls apart as a result. The treatment of BR by RNase H, Nt.AlwI and Nt.BspQI is conducted in the presence of an assisting DNA strand complementary to the corresponding ssRNA or ssDNA ring. LX and CX represent X-nt linear and circular species, respectively. In all the gels, lane M contains the DNA size markers.
Article Snippet: Digestion with various nucleases Various nucleases were used in this work, including Nt.AlwI (NEB), Nt.BspQI (NEB), RNase H (NEB), DNase I (NEB) and RNase R (Epicentre).
Techniques: Ligation, Sequencing, Construct, Purification