Entwicklung Aptamer-basierter RNA Markierungstechniken und Untersuchung der RNA Dynamik innerhalb lebener Zellen mittels FRET
Biochemie
Zellbiologie
Zusammenfassung der Projektergebnisse
The original research proposal dealt with 2 main goals, (1) To develop RNA mimics of red fluorescent proteins for multiplexed RNA imaging and (2) to develop a simplified approach to image RNA-protein interactions in living cells, over a period of two years. Due to a reduction of funding to one year the main focus of the presented work was laid on the development of new and photostable RNA mimics of red fluorescent proteins. By joining the Jaffrey lab the Spinach aptamer and the further optimized Broccoli aptamer were the already established RNA mimics of fluorescent proteins. Both aptamers can induce green fluorescence upon binding a non-fluorescent fluorogen, DFHBI. DFHBI mimics the fluorophore of green fluorescent protein and is colorless in aqueous solution. Upon binding to Spinach or Broccoli green fluorescence can be observed. Based on the DFHBI molecule the Jaffrey lab synthesized an analog to the red fluorescent protein, DFHO. DFHO was used in classical SELEX approach and resulted in a yellow fluorescing aptamer which was finally named corn. Alternative, non-fluorescent protein derived dyes such as α-CN-TO and azetidine modified dyes were expected to show better photophysical properties than the already published DFHBI. Unlike DFHBI and DFHO, CN-TO and the azetidine modified DFHBI fluorophore had no linker attached where the dye could be coupled to a resin. Therefore no standard SELEX, where the target is immobilized on a resin and aptamers can be selected by washing away low affinity binding aptamers leaving high affinity binding species on the resin, could be performed. All the dyes described here were synthesized by a co-worker Wenjiao Song. Do to this lack of a linker, alternative SELEX methods were investigated. Namely structure switching approaches where not the target but the library is immobilized. This approach would allow to select for all different types of small molecules that can’t be attached to a resin due to lack of a linker. After addition of the target the binding aptamers are released while non-binding structures remain immobilized. Two different SELEX strategies were tested, namely a bead based capture approach and a graphene oxide based approach. Unfortunately both approaches didn’t result in new species of fluorescence inducing aptamers. Besides the search for a new method of finding red fluorescent aptamers the optimization of Corn and photophysical characterization became a major goal and resulted in deep insight in the architecture of the corn aptamer, which is in contrast to Spinach and Broccoli a dimer of two identical aptamers.
Projektbezogene Publikationen (Auswahl)
- (2017) Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nature chemical biology 13 (11) 1187–1194
Song, Wenjiao; Filonov, Grigory S.; Kim, Hyaeyeong; Hirsch, Markus; Li, Xing; Moon, Jared D.; Jaffrey, Samie R.
(Siehe online unter https://doi.org/10.1038/nchembio.2477)
