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Summary
Biosensors have been successfully applied to study DNA hybridization reactions for several years. However, this detection principle has only rarely been used to investigate DNA processing enzymes. In the present work, the versatility of surface analytical tools has been demonstrated not only for monitoring biomolecular recognition events but also for measuring the catalytic activity of an enzyme. The study was centered around the replication of surface-tethered oligonucleotides using a bacterial DNA polymerase, the Klenow fragment. The replication process could be successfully monitored in real-time utilizing Surface Plasmon enhanced Fluorescence Spectroscopy (SPFS) and the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D).
Since both techniques require functionalization of a gold surface, identical surface architectures were used as matrix for DNA immobilization. Biotinylated primer oligonucleotides were bound to a streptavidin arrangement that was tethered to the gold surface by a self-assembled monolayer of biotinylated thiols. A DNA substrate with recessed 3’-terminus was formed by hybridization of the primer to an unmodified template oligonucleotide. The stepwise immobilization of the matrix components was followed using both, SPFS (label-free) and QCM-D, providing control over the multi-layer system. By comparison of the results obtained from both techniques, it was possible to determine the water content of each layer. In agreement with previous studies, streptavidin was found to form a monomolecular film exhibiting 50 % water as reported for 2-dimensional streptavidin crystals. A water content of 90 % was measured for the DNA films irrespective of the length of the primer oligonucleotides. Thus, identical amounts of water are coupled per nucleotide leading to a film with a high lateral dilution. As predicted for QCM measurements in liquids, the water coupled due to viscous drag was sensed as additional mass, which made the technique by a factor of ~10 more sensitive for the detection of DNA than SPR. Analysis of the QCM-D data using a viscoelastic model revealed that streptavidin forms a very rigid film, whereas the DNA films are relatively soft provoking large dissipative losses. Accordingly, one can assume elongated DNA chains, which are only moderately coiled. Only sequences being shorter than 15 nucleotides were significantly stiffer than longer oligonucleotides.
The sensor surface could be shown to be well-suited for DNA polymerase action. Binding and catalytic constants derived for enzymatic DNA synthesis were in good agreement with results obtained from solution. Thus, the catalytic activity of the Klenow fragment was not perturbed by the proximity to the surface. This was also proven by introducing a spacer region that increased the distance between enzyme and surface, which did not influence the experimental results. By introducing a poly-thymine tail as spacer, a second putative binding site for DNA polymerases was created. However, identical amounts of enzyme bound to the 15 nucleotide long primer/template duplex of the different DNA substrates, demonstrating that exclusively 1:1 polymerase/DNA complexes were formed.
QCM-D and SPFS differed in their capability to resolve different steps involved in DNA replication. SPR was incapable of sensing the mass increase due to DNA synthesis. Therefore, DNA synthesis was visualized by incorporation of Cy5-labeled dCTP into the DNA strand. This way, enzyme binding and release (accounted for by reflectivity), and DNA polymerization (fluorescence intensity) were obtained as well separated signals which facilitated the interpretation of the experimental curves. Deviating from recent findings, the polymerase was clearly found to prefer the natural unlabeled dCTP substrate over the labeled one.
In contrast, the mass increase during DNA polymerization could be easily detected by QCM-D. Since the replication of immobilized DNA cannot occur synchronously at the surface, the signals being attributed to a mass loss due to enzyme release and an increase due to DNA synthesis were found to be superimposed. Despite of this complication, the process could be interpreted based on changes in shear viscosity. This became possible because formation of the double stranded replication product and the polymerase/DNA complex had opposite effects on the stiffness of the DNA films: while an identical viscosity was calculated before and after elongation, it was significantly increased as long as the polymerase directly interacted with the oligonucleotides. The latter finding is attributed to a loss of motional freedom and changes in the chemical environment of the oligonucleotides in complex with the DNA polymerase.
In conclusion, both techniques, QCM-D and SPFS, were successfully applied as biosensors for the real-time analysis of DNA replication. However, it makes sense to use them as complementary methods addressing different objectives. SPFS is unbeaten in sensitivity for the manipulation of single nucleotides; even dye concentrations of ~5 fmol/cm2 could be easily detected. The strength of QCM-D lies clearly in its ability to give insights into the conformational dynamics of the DNA film.
