Evaluation of ligation methods and the synthesis of a specific PNA-encoded peptide library
Stindl, Martin Maria Matthias
Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.