Ebbe Sloth Andersen

Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells. We introduce an architecture for designing artificial RNA structures that fold from a single strand, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern. We constructed RNA tiles that assemble into hexagonal lattices and demonstrated that lattices can be made by annealing and/or cotranscriptional folding. Tiles can be scaled up to 660 nucleotides in length, reaching a size comparable to that of large natural ribozymes.

The DNA origami method allows the folding of long, single-stranded DNA sequences into arbitrary two-dimensional structures by a set of designed oligonucleotides. The method has revealed an unexpected strength and efficiency for programmed self-assembly of molecular nanostructures and makes it possible to produce fully addressable nanostructures with wide-reaching application potential within the emerging area of nanoscience. Here we present a user-friendly software package for designing DNA origami structures ( http://www.cdna.dk/origami ) and demonstrate its use by the design of a dolphin-like DNA origami structure that was imaged by high-resolution AFM in liquid. The software package provides automatic generation of DNA origami structures, manual editing, interactive overviews, atomic models, tracks the design history, and has a fully extendable toolbox. From the AFM images, it was demonstrated that different designs of the dolphin tail region provided various levels of flexibility in a predictable fashion. Finally, we show that the addition of specific attachment sites promotes dimerization between two independently self-assembled dolphin structures, and that these interactions stabilize the flexible tail.

Biological systems use compartmentalisation as a general strategy to control enzymatic reactions by precisely regulating enzyme-substrate interactions. With the advent of DNA nanotechnology, it has become possible to rationally design DNA-based nano-containers with programmable structural and dynamic properties. These DNA nanostructures have been used to cage enzymes, but control over enzyme-substrate interactions using a dynamic DNA nanostructure has not been achieved yet. Here we introduce a DNA origami device that functions as a nanoscale vault: an enzyme is loaded in an isolated cavity and the access to free substrate molecules is controlled by a multi-lock mechanism. The DNA vault is characterised for features such as reversible opening/closing, cargo loading and wall porosity, and is shown to control the enzymatic reaction catalysed by an encapsulated protease. The DNA vault represents a general concept to control enzyme-substrate interactions by inducing conformational changes in a rationally designed DNA nanodevice.

Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.