DNA ORIGAMI – Folding of the Vector (pCDH–CMV–MCS–EF1–puro) into a Predefined Shape Using 18, 20mer Staples
DNA origami is the process in which long single-stranded DNA molecules are folded into arbitrary planar nanostructures with the aid of many short staple strands. DNA origami can be used to construct not only arbitrary two-dimensional nanostructures but also nano-sized breadboards for the arraying of nanomaterials or even complicated three-dimensional nano-objects.
The present project aims to fold the DNA in the shape of a bell.Double stranded DNA (pCDH–CMV–MCS–EF1–puro, 7.384Kb size) was used as a scaffold which is isolated from DH5- α E coli clone. Miniprep and Maxi prep were performed in order prepare sufficient amount of scaffold (Ref). Agarose Gel Electrophoresis and Spectrophotometric analysis (EppendorfBiophotometer). The circularly permuted plasmid was restricted by unique enzymes- Kpn I, Xba I and Not I to generate a linear plasmid. The band representing the linear plasmid was gel eluted using the Qiagen gel elution kit. Eighteen, 20mer staples were designed and custom made from Sigma which are unique in its sequence and produced no dimmers with any of the other staples. Folding was performed when 10nM of scaffold strands were mixed with 50nM oligonucleotide staple (1:5 ratio) strands by carrying out a single pot PCR reaction. The formation of the structure was visualised through Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM).