Advanced DNA-based Molecular Tools for Cell Signalling Analysis
UCD Systems Biology Ireland are delighted to welcome Professor Ola Söderberg (Uppsala University, Sweden) who will present a guest lecture on advanced DNA-based molecular tools for analysis of cell signalling on Friday 4th November 2016 at 10am in the UCD Conway Institute Lecture Theatre. Note venue has changed.
About Professor Ola Söderberg’s Research
High-resolution analysis of proteins or protein complexes requires either advanced microscopy, or molecular methods that combine distance dependence with signal amplification. By using DNA molecules as a tool in such molecular methods one can predict hybridization between DNA molecules and use the vast variety of enzymes to modify and amplify the DNA molecules used in the method.
One such method is Proximity Ligation Assay (PLA) where pairs of antibodies equipped with DNA oligonucleotides (so-called proximity probes) are used to target proteins. Proximal binding of such probes template the creation of a circular DNA molecule, which can be amplified using rolling circle amplification (RCA). The single-stranded RCA product from a single recognition event will contain several hundreds of repetitive motifs that can be visualized by hybridization of fluorophore-conjugated oligonucleotides.
The distance requirement for the formation of the DNA circles will be dependent on the size of the affinity reagents and the length and polarity of the oligonucleotides used, ranging from a few nm up to several tens of nm. However, the RCA product will be much larger than the distance between the targeted epitopes, up to a micron in diameter.
To provide an increased dynamic range, alternative designs may be used to compact the RCA products, or to generate different species of products that can be detected with unique fluorophores. An alternative to PLA would be the proximity-dependent initiation of hybridization chain reactions (proxHCR) where the proximity probes consists of DNA hairpins.
The addition of an activator oligonucleotide that can hybridize to one of the hairpins will liberate a sequence motif that will invade the second species, if they are in close proximity. The now bridged proximity probes reveal the initiator motif that will prime a hybridization chain reaction of fluorophore-labelled hairpins, creating a long fluorophore-labelled double-stranded DNA molecule.
Prof Söderberg will describe different approaches to use DNA-based molecular tools for analysis of cell signalling and will outline what advantages the different variants provide.
All academic and research staff are welcome.