DNA Microarray technologies & PNA

This project includes the use of DNA microarray technologies which has allowed them to interface DNA arrays with combinatorial libraries. PNA-encoded libraries were developed and successful used to study kinases and proteases for the very first time by Juanjo and Laurent Bialy, an important mention goes to Lise Keinicke. Currently 10,000 member libraries are being tested in order to profile different enzymes while other PNA-encoded strategies are being developed. One of the ongoing projects is the use of cell-based microarrays. Following the successful application of reverse transfection microarrays to study transfection agent libraries and the experience achieved by the Bradley group in the development of polymer arrays , collaborations with Tim Elliot and Salim Khakoo are underway to exploit the huge potential of this technology.

Figure 1. PNA-encoded libraries. From solution assays to 2D microarrays.

Figure 2. Hybridization of a 10,000 member PNA-encoded library using a custom OGT DNA microarray.

Figure 3. Cells adhered onto a microarray spot. Different detection systems can be used such as fluorescently labelled antibodies.

Peptide Nucleic Acid (PNA): Novel Applications in HTS

PNA are well-known DNA mimetics which hybridise complementary DNA sequences with higher affinity than their DNA counter-parts. While DNA has a sugar-phosphonate backbone, PNA has a 2-aminoethylglycinate one.

The synthesis of new protected PNA monomers has been developed, thus allowing the solid-phase synthesis of various PNA conjugates. The aim of the project involves the use of these conjugates in a wide range of applications, for example antisense and antigene bio-assays and High Throughput Screening (HTS) by utilising DNA microarray technology. The Group has acquired the complete equipment to make and analyse its own DNA or PNA chips. This equipment includes a Genetix QArrayMini Microarray and a LaVision BioTech Bioanalyzer 4F/4S Scanner (white-light source coupled to a CCD camera).

High density OGT custom DNA microarray hybridised with a synthetic PNA encoded peptide library (10,000 members)

DNA/PNA-Microarray for Receptor-targeting

The ability to screen and identify new ligands for cell surface receptors has long been a goal of ours and many other research groups and organisations. Principally it would allow targeting of receptors, such as integrins and G-protein coupled receptors, which currently are amongst the most heavily investigated drug targets in the pharmaceutical industry. In our group screening for new peptide ligands in a high-throughput assay against live cells has been achieved with the split-and-mix synthesis and incubation of a 10,000 member PNA-encoded peptide library with cells over-expressing the avb5 and avb3 integrins (D54) or CCR6 (HEK293T-CCR6). Microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new cell specific ligands for avb3 and avb5 integrins and CCR6 and offers an approach to ligand discovery that allows the comparative, competitive and simultaneous analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between cell types. This approach establishes a strategy for high-throughput screening for the identification of ligands for integrins and GPCRs offering an efficient approach to de-orphanise the many GPCRs, for which there are no known ligands and thereby identify new drug-targets as well as leads.

DNA/PNA-Microarray for Tumor-targeting Peptides

The ability to target tumours or develop organ-biased therapies with a reduction of off-target effects has been long a long-term goal of ours as well as many other research groups and pharmaceutical organisations, and indeed tumour homing peptides have entered clinical trials. However, this research area is lacking high-throughput methods that allow screening for peptides homing to any tissue of interest. In this aim we synthesized a PNA-encoded 1296 member peptide library and incubated it with 9 human cell types. Cell-penetrating library members were extracted and hybridized onto DNA microarrays and the peptide identity determined via deconvolution. This revealed a highly acidic tetrapeptide as a global consensus sequence, a surprise in view of the basic residues typically observed in cell penetrating peptides. This tetrapeptide revealed cellular uptake comparable to a known basic peptide. Clustering analysis identified peptides showing clear cellular specificity, this was verified by peptide synthesis and uptake analysis by flow-cytometry and confocal microscopy. This approach establishes a general strategy for the identification of small peptides as homing peptides that allow selective delivery into any cell type or tissue of interest, such as a targeted organ or a tumour.