Thomas Ljungdahl

Membrane-anchored DNA assembly for energy and electron transfer.

In this work we examine the trapping and conversion of visible light energy into chemical energy using a supramolecular assembly. The assembly consists of a light-absorbing antenna and a porphyrin redox center, which are covalently attached to two complementary 14-mer DNA strands, hybridized to form a double helix and anchored to a lipid membrane. The excitation energy is finally trapped in the lipid phase of the membrane as a benzoquinone radical anion that could potentially be used in subsequent chemical reactions. In addition, in this model complex, the hydrophobic porphyrin moiety acts as an anchor into the liposome positioning the DNA construct on the lipid membrane surface. The results show the suitability of our system as a prototype for DNA-based light-harvesting devices, in which energy transfer from the aqueous phase to the interior of the lipid membrane is followed by charge separation.

Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption.

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.

Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast

Abstract Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Dinér et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed.

Positional Scanning Peptide Libraries for Kinase Substrate Specificity Determinations: Straightforward and Reproducible Synthesis Using Pentafluorophenyl Esters.

An efficient method to synthesize positional scanning synthetic combinatorial libraries (PS-SCLs) for studying the specificity of protein kinases is presented. Isokinetic ratios for pentafluorophenyl esters were determined iteratively using a new approach incorporating high performance liquid chromatography (HPLC) quantification and statistical experimental design. In the development process a large amount of work was put in to find efficient ways of screening for new isokinetic mixtures and to optimize the process of PS-SCL synthesis. The newly developed methods for the screening of isokinetic mixtures could be used for the screening of other interesting mixtures, but more importantly, the isokinetic ratios determined for the preactivated pentafluorophenyl esters were incorporated into a new efficient protocol. This straightforward protocol allows for a convenient synthesis of high quality PS-SCLs regardless of previous experience in solid phase synthesis.

A Membrane Anchored DNA-based Energy/Electron Transfer Assembly

In this work the trapping and conversion of visible light energy into chemical energy is examined using a supramolecular assembly. This consists of a light absorbing antenna and a porphyrin redox centre both covalently attached to a DNA strand, which in turn is bound to a lipid membrane. The excitation energy is finally trapped as a benzoquinone radical anion that could potentially be used in subsequent chemical reactions.