AbstractsBiology & Animal Science

Novel luminescent peptide conjugates for assessing protein and other biomolecules location and function

by Lorraine Blackmore

Institution: Dublin City University
Department: School of Chemical Sciences
Year: 2013
Keywords: Chemistry; Biosensors; Peptide synethesis; ruthenium polypyridyl complexes; live cell imaging; protein; nuclear localisation signals; maganin 2; BID peptides
Record ID: 1180913
Full text PDF: http://doras.dcu.ie/17714/


Luminescent dye molecules capable of passive cell membrane transport may be used as molecular probes for example in cellular imaging. Ru(II) polypyridul complexes have been under extensive investigation for many years in supramolecular assemblies in applications ranging from sensors to photovoltaics. This is due to very their favourable photophysical and redox properties including; absorbance and emission in the visible region of the spectrum, large stokes shifts, long lifetimes, intense luminescence, good photostability and useful photosensitising properties for photodynamic therapy. Despite these properties, there has until very recently been very little examination of the potential of such complexes in cell imaging, which has mainly been due to the poor uptake of such complexes by the living cell. However, our group recently reported that polyarginine labelled ruthenium complexes were efficiently and rapidly transported across the cell membrane into the cytoplasm. Such chromophores provide unique opportunities for providing dynamic insigts into environment in living cells avoiding limitations associated with fixation. This thesis continues this work by reporting on the synthesis, purification, characterisation and optical properties of novel supramolecular bioconjugates comprised of one of two luminescent Ru(II) polypyridyl complexes bridged through an aliphatic linker to a range of synthetic peptides aimed towards transport and direction of these luminophores or derived from apoptotic proteins. In the former case, preliminary studies of their uptake, localisation and cytotoxicity toward mammalian cells is also presented. Chapter 1 introduces the topic and describes how metal complexes have been used previously in cellular imaging. It also describes how peptide conjugation can lead to targeted delivery for improved applications in medical diagnostics, photodynamic therapy, cellular imaging and pharmaceutical drug delivery. Chapter 2, describes the experimental methods used. In chapter 3, the synthesis, purification and characterisation of six novel dye conjugates to nuclear localising peptides is described. The influence of metal complex, bridge and peptide on the optical properties and on the uptake of these probes is described. The complexes exhibit long lived, intense, and oxygen sensitive luminescence which are relatively unchanged in the conjugates from the parent dies. Preliminary results on their application in cell imaging are also presented which demonstrate excellent nuclear permeability and nuclear localisation which depends on the hydrophobicity of the metal centre. Chapter 4 describes how the sequence of the BID protein, a pro apoptotic member of the BCL-2 family, was prepared using solid phase peptide synthesis as a number of overlapping peptides. The objective was to identify a functional region of the protein. Two BID sequences were selected for conjugation to probes for localisation and function studies. Along with the BID sequences, another BCL-2 family member was investigated.…