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

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