Centre for Biological Signalling Studies SELEX for synthetic biology and nanoscale applications

Description of the project:
SELEX for synthetic biology and nanoscale applications Emerging new techniques such as super-resolution microscopy, hold the promise of detecting nanostructures of cell surface receptor, thus serving as invaluable tools for deciphering the receptor signaling mechanism. However, these methods are often limited by the availability of labeling tools and the general low labeling efficiency. Remarkably, aptamers, single-stranded DNA or RNA oligonucleotides selected in vitro for the desired targets, have been shown to surpass the normal antibody in reaching a higher labeling density for super-resolution microscopy. Although aptamers can be easily selected against virtually any target through an in vitro selection process known as SELEX (systematic evolution of ligands by exponential enrichment), the traditional random cloning and sequencing approaches only enable sampling of a small portion of the enriched sequences. For the determination of a small number of high affinity binders, it is often needed to perform the SELEX with more than 20 rounds, which is expensive and time consuming. Even worse, SELEX is a black box in this setting to be opened only at the last round. This creates many problems such as low reproducibility (different runs will produce different aptamers, sharing no similarity), PCR artifacts or background binding. In addition, the current technology usually does not allow inferring more general sequential or structural properties of high-affinity binding RNA aptamers. This hinders their applicability for synthetic biology applications, where an aptamer is integrated in a longer transcript to combine several functions. The rational design of these molecules would require such properties to be checked. To solve these problems, we propose to 1) combine the in vitro SELEX power with next generation sequencing (NGS) and advanced bioinformatics analysis to detect short binding motifs and their sequential and structural properties, and 2) to improve the SELEX protocol by partially including the iCLIP protocol, which is used to detect binding sites of RNA-binding proteins. The later will allow us to experimentally determine likely binding regions, a so far unsolved problem in SELEX. In addition, the project requires a constant interplay between lab work and bioinformatics, where selected motifs are tested in the lab, giving rise to additional information about functional motifs that will be used to improve the bioinformatics analysis.

Additional information: http://www.bioss.uni-freiburg.de
Phone: +49 (0)761 203 7461
Email: backofen@informatik.uni-freiburg.de
Runtime:
Start of project: 01.11.2015
End of project: 31.10.2017
Project Management:
Albert-Ludwigs-University Freiburg
Prof. Dr. Rolf Backofen
Bioinformatik
Prof. Dr. Rolf Backofen
Georges-Köhler-Allee 106
79110 Freiburg
Germany

Phone: +49 (0) 761-203-7461
Fax: +49 (0) 761-203-7462
Email: backofen@informatik.uni-freiburg.de
http://www.bioinf.uni-freiburg.de
Actual Research Report
Financing:
  • Exzellenzinitiative DFG
Keywords:
    BIOSS 2