Prof W S James
Dr N Janjic
No more applications being accepted
Funded PhD Project (European/UK Students Only)
Induced pluripotent stem cells have unprecedented biotechnological potential, from providing physiological models for the identification of pharmaceutical leads to the production of defined tissues for cell therapy. A key barrier to the large scale industrial exploitation of these potentials stems from the two-dimensional, demanding culture format and the cost and irreproducibility of the biological additives to culture media. These challenges could be addressed by the modulation of stem cell behaviour in vitro by ligation of their cell-surface receptors by high-affinity nucleic acid ligands. SomaLogic has developed such aptamers using their proprietary SOMAmer technology, and this project will build on a research collaboration framework agreement recently concluded between them and Oxford to evaluate their potential. The student would benefit from the support of two other collaborative projects currently underway between the two groups.
Nucleic acid libraries offer a rich source of ligands that can be efficiently selected by in vitro evolution from randomized libraries of enormous size. The numerical advantage of starting libraries (>1015 sequences) is offset by the more limited chemical diversity of nucleic acids compared with proteins. We have focused on bridging this diversity gap by introducing functional groups absent in natural nucleic acid libraries, but common in protein-protein contacts or interactions between small molecules and their protein targets. The 5-position of pyrimidines is a convenient point of attachment for a variety of functional groups through a conformationally-restricted amide linkage. The expanded chemical diversity “front loaded” into the starting libraries results in higher success rate of SELEX, expanded range of accessible epitopes, and enhancement in nuclease resistance. Among the 5-position side chains we tested, hydrophobic aromatic moieties typically yield best ligands. Not surprisingly, amino acids like tyrosine, tryptophan and phenylalanine are overrepresented in antigen recognition regions of antibodies. Post-SELEX optimization can be readily achieved with a library of alternative side chains that spans a range of physicochemical properties, in an exercise resembling SAR optimization of small molecules, or affinity maturation in antibodies. We named this new generation of nucleic acid ligands slow off-rate modified aptamers, or SOMAmers. Based on co-crystal structures of SOMAmer-protein complexes, these modifications create entirely new structural motifs and generate extensive hydrophobic binding surfaces, in contrast to mainly polar surfaces observed with conventional aptamers. Improved starting libraries and selection methods have allowed us to build a collection of over 3,000 SOMAmers. This set of novel binding reagents enables high-content proteomics, biomarker discovery and the development of novel diagnostic and therapeutic agents.
In the first instance, the student would investigate the utility of SOMAmers already generated by SomaLogic to the cell surface adhesion molecule, e-Cadherin – one of over 1,000 targets in the library. In a separate collaboration with Dr Angela Russell of the Oxford Chemistry Department, supported by the iMi StemBancc programme, JMSCF have confirmed in human Pluripotent Stem Cells the previously published work with mouse ES cells showing that antibodies to e-Cadherin can substitute for homotypic interactions between stem cells, allowing them to be cultivated in suspension, and are evaluating the potential for cyclic peptides to substitute for the antibodies.
In pilot experiments, we have recently demonstrated that a subset of six, previously synthesized Somamers to e-cadherin can inhibit the formation of adherens junctions in monolayers of iPS cells in vitro (see figure).
Accordingly, the student will advance these studies by:
• mapping the binding sites of Somamers to eCadherin using SPR technology
• evaluate the effect of multimerizing the 5’-amine Somamers on their antagonist activity
• Evaluate a large panel of new Somamers of alternate chemistries currently being produced in Boulder.
Depending on progress, the student would have the opportunity to evaluate Somamers against other cell surface molecules involved in stem cell maintenance and differentiation.
Importantly, the student would also use the unique SOMAmer reagents to explore the biology adherens junctions in the physiology of stem cells. Using a combination of confocal microscopy and proteomic techniques in use in the James Lab, they would explore the kinetics of the formation of adherens junctions in both primed and naïve pluripotent stem cells, and the effect of their disruption by Somamers to e-Cadherin on down-stream signalling pathways. The results would not only shed critical light on the maintenance of the pluripotent states, but also offer further avenues for biotechnological manipulation.
Principal Location: Sir William Dunn School of Pathology, University of Oxford
Non-academic partner location: SomaLogic is located in a well-designed facility in Boulder, Colorado and has about 170 employees. The facility has approximately 20,000 square feet of laboratory space, separated into two buildings for technology development, aptamer screening, assay development and assay execution, and chemistry. Training in the latest methods of nucleic acid chemistry and biotechnology, including the use of aptamers in high-sensitivity proteome detection arrays will be provided during the residency at SomaLogic.
This project is supported through the Oxford Interdisciplinary Bioscience Doctoral Training Partnership (DTP) BBSRC Industrial CASE (iCASE) studentship programme. The student recruited to this project will join a cohort of students enrolled in the DTP’s interdisciplinary training programme, and will be able to take full advantage of the training and networking opportunities available through the DTP. For further details please visit www.biodtp.ox.ac.uk.
Prospective applicants should contact the project supervisor Professor William James ([Email Address Removed]) prior to submitting an application.
Applications for this project will be made via the Oxford Interdisciplinary Bioscience DTP. For further details please visit www.biodtp.ox.ac.uk.
Attributes of suitable applicants:
A first (bachelor’s) degree in Biochemistry, Cell Biology, Chemistry or related disciplines is essential. Evidence that the applicant has productive experience of at least one substantial, supervised research project in these fields is essential, either as a component of a formal degree curriculum, or otherwise. Paid work experience or internships in industrial or academic labs is desirable.
First-hand experience of nucleic acid chemistry and tissue culture is desirable but not essential.
A disposition to think about cellular systems in both a physicochemical and a biological manner is essential, as is a willingness to learn, diligence and good organizational skills.
This project is funded for four years by the Biotechnology and Biological Sciences Research Council BBSRC. BBSRC eligibility criteria apply (http://www.bbsrc.ac.uk/documents/studentship-eligibility-pdf/). EU nationals who do not meet BBSRC residence criteria are encouraged to contact the programme administrator to check their eligibility for BBSRC funding before submitting a formal application. Successful students will receive a stipend of no less than the standard RCUK stipend rate, currently set at £14,296 per year, which will usually be supplemented by the industrial partner.
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