The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form). We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role.

The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study. We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results. Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.

The mechanical properties of cells are important for many biological processes, including wound healing, cancers, and embryogenesis. Currently, our understanding of cell mechanical properties remains incomplete. Different techniques have been used to probe different aspects of the mechanical properties of cells, among them microplate rheology, optical tweezers, micropipette aspiration, and magnetic twisting cytometry. These techniques have given rise to different theoretical descriptions, reaching from simple Kelvin-Voigt or Maxwell models to fractional such as power law models, and their combinations. Atomic force microscopy (AFM) is a flexible technique that enables global and local probing of adherent cells. Here, using an AFM, we indented single retinal pigmented epithelium cells adhering to the bottom of a culture dish. The indentation was performed at two locations: above the nucleus, and towards the periphery of the cell. We applied creep compliance, stress relaxation, and oscillatory rheological tests to wild type and drug modified cells. Considering known fractional and semi-fractional descriptions, we found the extracted parameters to correlate. Moreover, the Young’s modulus as obtained from the initial indentation strongly correlated with all of the parameters from the applied power-law descriptions. Our study shows that the results from different rheological tests are directly comparable. This can be used in the future, for example, to reduce the number of measurements in planned experiments. Apparently, under these experimental conditions, the cells possess a limited number of degrees of freedom as their rheological properties change.