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Laboratory of Molecular Biophysics
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Previous: Polo-like kinase (Plk1), Next: Acknowledgments and References, Up: Protein Kinases, Return to: Contents.
Details of our electron microscope studies on a number of protein single particle complexes and on 2D arrays are described in section 11 (Dr Catherine Vénien-Bryan). Here we described a new approach in an attempt to provide methods of organising specimens for EM observation. We aim to produce a periodic DNA template that will order proteins and protein complexes. DNA is a versatile material in which the highly specific molecular recognition between complementary oligonucleotides offers several unusual applications. DNA has been exploited in DNA computation, in self-assembly systems that use DNA as intelligent glues, and as a molecular machine in the form of tweezers in which DNA is used not only as a structural material but also as a fuel (Yurke et al. 2000). Winfree et al. (Winfree et al. 1998) have made 2D periodic DNA structures and have imaged them by atomic force microscopy. Following this work, Dr Andrew Turberfield and James Mitchell (Clarendon Laboratory) have designed oligonucleotides that self assemble into approximately oblong rigid tiles based on a double crossover motif incorporating two Holliday junctions. Two types of tile were prepared, each with four unhybridised 6-base sticky ends at its corner. When the two species of tile are annealed together, hybridisation of the sticky ends links them in a periodic 2D array. One oligo tile was labelled with biotin at the 5' end.
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We have examined these structures in the Phillips CM120 electron microscope in the Laboratory. The best results were obtained by spreading the sample across holey carbon/perforated films using negative staining methods with 5% ammonium molybdate, 0.1% trehalose, and pH 7.0 (Harris and Scheffler 2002). The specimens reveal a ribbon-like structure with no obvious periodicity. When the biotin labelled oligos are decorated with streptavidin, a linear periodicity of approximately 27.7 nm is apparent with the streptavidin molecules bound across the width of the DNA ribbon (Figure 7). The spacing is roughly consistent with the periodicity expected for the DNA scaffold. The diffraction pattern of the micrograph extends to the 5th order corresponding to a spacing of 5.4 nm. Clearly there is some way to go before we have a scaffold that will allow production of ordered arrays of proteins that can be imaged at high resolution. The pilot study has established the feasibility of producing regular labelled DNA scaffolds. The work will be pursued to produce new scaffolds, to use new labelling techniques such as glutathione to allow a wide range of proteins to be bound as GST fusions, and to explore the structures of selected DNA binding proteins with oligos designed with sequence specific motifs.
Previous: Polo-like kinase (Plk1), Next: Acknowledgments and References, Up: Protein Kinases, Return to: Contents.