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.

Protein bioconjugates are in high demand for applications in biomedicine, diagnostics, chemical biology and bionanotechnology. Proteins are large and sensitive molecules containing multiple different functional groups and in particular nucleophilic groups. In bioconjugation reactions it can therefore be challenging to obtain a homogeneous product in high yield. Numerous strategies for protein conjugation have been developed, of which a vast majority target lysine, cysteine and to a lesser extend tyrosine. Likewise, several methods that involve recombinantly engineered protein tags have been reported. In recent years a number of methods have emerged for chemical bioconjugation to other amino acids and in this review, we present the progress in this area.

The development of methods for conjugation of DNAt op roteins is of high relevance for the integration of protein function and DNAs tructures.H ere,w ed emonstrate that protein-binding peptides can direct aD NA-templated reaction, selectively furnishing DNA-protein conjugates with one DNAlabel. Quantitative conversion of oligonucleotides is achieved at lows toichiometries and the reaction can be performed in complex biological matrixes,such as cell lysates. Further,w eh ave used as tar-like pentameric DNAn anostructure to assemble five DNA-Rituximab conjugates,m ade by our reported method, into apseudo-IgM antibody structure that was subsequently characterized by negative-stain transmission electron microscopy( nsTEM) analysis.

Site-directed addition of a single thiols handle to proteins by means of temporary disulphide rebridging of solvent exposed disulphides is obtained with a new labelling reagent.